Nucleoside derivative or salt thereof, polynucleotide synthesis reagent, method for producing polynucleotide, polynucleotide, and method for producing binding nucleic acid molecule

ABSTRACT

The present invention provides a novel nucleoside derivative or a salt thereof, a polynucleotide synthesis reagent, a method for producing a polynucleotide, a polynucleotide, and a method for producing a binding nucleic acid molecule. The nucleoside derivative or a salt thereof of the present invention is represented by the following chemical formula (1): 
     
       
         
         
             
             
         
       
     
     where in the chemical formula (1), Su is an atomic group having a sugar skeleton at a nucleoside residue or an atomic group having a sugar phosphate skeleton at a nucleotide residue, and may or may not have a protecting group, L 1  and L 2  are each independently a straight-chain or branched, saturated or unsaturated hydrocarbon group having 2 to 10 carbon atoms, X 1  and X 2  are each independently an imino group (—NR 1 —), an ether group (—O—), or a thioether group (—S—), and the R 1  is a hydrogen atom or a straight-chain or branched, saturated or unsaturated hydrocarbon group having 2 to 10 carbon atoms.

TECHNICAL FIELD

The present invention relates to a nucleoside derivative or a saltthereof, a polynucleotide synthesis reagent, a method for producing apolynucleotide, a polynucleotide, and a method for producing a bindingnucleic acid molecule.

BACKGROUND ART

In order to analyze a target in a specimen, a binding molecule thatbinds to a target is used. In addition to an antibody, a binding nucleicacid molecule that binds to a target such as an aptamer is also used asa binding molecule that binds to the target (Patent Literature 1).

As a method for obtaining the binding nucleic acid molecule, a SELEX(Systematic Evolution of Ligands by Exponential Enrichment) method inwhich a target is caused to come into contact with a large number ofcandidate polynucleotides and a polynucleotide that binds to the targetamong the candidate polynucleotides is selected as the binding nucleicacid molecule is known. When a binding nucleic acid molecule is obtainedby the SELEX method, a modified nucleoside molecule obtained bymodifying a natural nucleoside molecule is also used in addition to anatural nucleoside molecule that constitutes the binding nucleic acidmolecule.

However, with known natural nucleosides and derivatives thereof, thereare targets for which binding nucleic acid molecules with sufficientbinding ability cannot be obtained. Therefore, there is a need formodified nucleoside derivatives that can be used, for example, in theproduction of aptamers.

CITATION LIST Patent Literature

Patent Literature 1: JP 2012-200204 A

SUMMARY OF INVENTION Technical Problem

Hence, the present invention is intended to provide a novel nucleosidederivative or a salt thereof, a polynucleotide synthesis reagent, amethod for producing a polynucleotide, a polynucleotide, and a methodfor producing a binding nucleic acid molecule.

Solution to Problem

The nucleoside derivative or a salt thereof of the present invention isrepresented by the following chemical formula (1).

in the chemical formula (1), Su is an atomic group having a sugarskeleton at a nucleoside residue or an atomic group having a sugarphosphate skeleton at a nucleotide residue, and may or may not have aprotecting group, L¹ and L² are each independently a straight-chain orbranched, saturated or unsaturated hydrocarbon group having 2 to 10carbon atoms, X¹ and X² are each independently an imino group (—NR¹—),an ether group (—O—), or a thioether group (—S—), and the R¹ is ahydrogen atom or a straight-chain or branched, saturated or unsaturatedhydrocarbon group having 2 to 10 carbon atoms.

The polynucleotide synthesis reagent of the present invention includes anucleotide derivative or a salt thereof including the nucleosidederivative or a salt thereof of the present invention.

The method for producing a polynucleotide of the present inventionincludes the step of synthesizing a polynucleotide using a nucleotidederivative or a salt thereof including the nucleoside derivative or asalt thereof of the present invention.

The polynucleotide of the present invention includes, as a buildingblock, a nucleotide derivative or a salt thereof including thenucleoside derivative or a salt thereof of the present invention.

The method for producing a binding nucleic acid molecule of the presentinvention includes the steps of: causing a candidate polynucleotide anda target to come into contact with each other; and selecting thecandidate polynucleotide bound to the target as a binding nucleic acidmolecule that binds to the target, and the candidate polynucleotide isthe polynucleotide of the present invention.

Advantageous Effects of Invention

The present invention can provide a novel nucleoside derivative or asalt thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the binding ability of an α-amylase-bindingnucleic acid molecule to α-amylase in Example 2.

FIG. 2 is a graph showing the binding ability of the secretoryimmunoglobulin A (sIgA)-binding nucleic acid molecule to sIgA in Example2.

FIG. 3 is a photograph showing the results of capillary electrophoresisin Example 2.

FIG. 4 is a photograph showing the results of the pull-down assay inExample 2.

FIGS. 5A to 5C are graphs showing the binding ability of the respectivetypes of β-defensin (BDN)4A-binding nucleic acid molecules to BDN4A inExample 3.

FIGS. 6A to 6C are graphs showing the binding ability of the respectivetypes of lysozyme-binding nucleic acid molecules to lysozyme in Example3.

FIG. 7 is a photograph showing the results of the pull-down assay inExample 3.

FIGS. 8A and 8B are photographs showing the results of the pull-downassay in Example 3.

FIGS. 9A and 9F are graphs showing the binding ability of the respectivetypes of α-amylase-binding nucleic acid molecules to α-amylase inExample 4.

FIG. 10 is a photograph showing the results of capillary electrophoresisin Example 4.

FIG. 11 is a photograph showing the results of the pull-down assay inExample 4.

FIGS. 12A to 12D are graphs showing the binding ability of therespective types of lactate dehydrogenase (LDH) 5-binding nucleic acidmolecules to LDHS in Example 5.

FIGS. 13A and 13B are graphs showing the binding ability of therespective types of interleukin (IL) 6-binding nucleic acid molecules toIL-6 in Example 5.

FIG. 14 is a graph showing the relative values of the binding amounts ofthe respective types of LDHS-binding nucleic acid molecules to LDHS inExample 5.

FIG. 15 is a graph showing the relative values of the binding amounts ofthe respective types of IL-6 binding nucleic acid molecules to IL-6 inExample 5.

FIG. 16 is a photograph showing the results of the pull-down assay inExample 5.

DESCRIPTION OF EMBODIMENTS

(Nucleoside Derivative or Salt Thereof)

The nucleoside derivative or a salt thereof of the present invention isrepresented by the following chemical formula (1), as mentioned above.

In the chemical formula (1), Su is an atomic group having a sugarskeleton at a nucleoside residue or an atomic group having a sugarphosphate skeleton at a nucleotide residue, and may or may not have aprotecting group, L¹ and L² are each independently a straight-chain orbranched, saturated or unsaturated hydrocarbon group having 2 to 10carbon atoms, X¹ and X² are each independently an imino group (—NR¹—),an ether group (—O—), or a thioether group (—S—), and the R¹ is ahydrogen atom or a straight-chain or branched, saturated or unsaturatedhydrocarbon group having 2 to 10 carbon atoms.

The nucleoside derivative of the present invention has two purinering-like structures. The nucleoside derivative of the present inventionthus has, for example, a relatively larger number of atoms capable ofinteracting within or between molecules than a nucleoside derivativehaving one purine ring-like structure. The binding nucleic acid moleculeincluding the nucleoside derivative of the present invention thereforehas an improved binding ability to a target, for example, compared to anucleoside derivative having one purine ring-like structure. Thus, withthe nucleoside derivative of the present invention, a binding nucleicacid molecule that exhibits excellent binding ability to a target can beproduced, for example.

In the chemical formula (1), L¹ and L² are each independently astraight-chain or branched, saturated or unsaturated hydrocarbon grouphaving 2 to 10 carbon atoms. The lower limit of the number of carbonatoms of L¹ is 2, the upper limit of the same is 10, preferably 8 or 6,and the range of the same is, for example, 2 to 8, 2 to 6. The number ofcarbon atoms of L¹ is preferably 2. The lower limit of the number ofcarbon atoms of L² is 2, the upper limit of the same is 10, preferably 8or 6, and the range of the same is, for example, 2 to 8, 2 to 6. Thenumber of carbon atoms of L² is preferably 2. Specific examples of L¹and L² include an ethylene group (—CH₂—CH₂—), a vinylene group(—CH═CH—), a propylene group (—CH₂—CH₂—CH₂—), an isopropylene group(—CH₂—CH(CH₃)−), a butylene group (—CH₂—CH₂—CH₂—CH₂—), a methylbutylenegroup (—CH₂—CH(CH₃)—CH₂—CH₂—), a dimethylbutylene group(—CH₂—CH(CH₃)—CH(CH₃)—CH₂—), an ethylbutylene group(—CH₂—CH(C₂H₅)—CH₂—CH₂—), a pentylene group (—CH₂—CH₂—CH₂—CH₂—CH₂—), ahexylene group (—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—), a heptylene group(—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—), and an octylene group(—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—). L¹ is preferably a vinylene group(—CH═CH—). L² is preferably an ethylene group (—CH₂—CH₂—). L¹ and L² maybe the same hydrocarbon group or different hydrocarbon groups. As aspecific example of the latter, L¹ is preferably a vinylene group(—CH═CH—), and L² is preferably an ethylene group (—CH₂—CH₂—).

In the chemical formula (1), X¹ and X² are each independently an iminogroup (—NR¹—), an ether group (—O—), or a thioether group (—S—). In theimino group, the R¹ is a hydrogen atom or a straight-chain or branched,saturated or unsaturated hydrocarbon group having 2 to 10 carbon atomsand is preferably a hydrogen atom. The description of L¹ and L² can beincorporated in the description of the straight-chain or branched,saturated or unsaturated hydrocarbon group having 2 to 10 carbon atomsby reference. X¹ is preferably an imino group (—NR¹—). X² is preferablyan imino group (—NR¹—). X¹ and X² may be the same substituent ordifferent substituents. As a specific example of the former, X¹ and X²is preferably an imino group (—NR¹—) and more preferably an NH group.

In the chemical formula (1), the atomic group having a sugar skeleton ata nucleoside residue is not particularly limited, and examples thereofinclude atomic groups having sugar skeletons on known natural orartificial nucleoside residues. Examples of the atomic group having asugar skeleton at a natural nucleoside residue include an atomic grouphaving a ribose skeleton at a ribonucleoside residue and an atomic grouphaving a deoxyribose skeleton on a deoxyribonucleoside. The atomic grouphaving a sugar skeleton at an artificial nucleoside residue can be, forexample, an atomic group having a bicyclic sugar skeleton at anartificial nucleoside residue, and specific examples thereof can be anatomic group having a ribose skeleton where an oxygen atom at2′-position and a carbon atom at 4′ position of ENA(2′-O,4′-C-Ethylene-bridged Nucleic Acids) or LNA (Locked Nucleic Acid)is crosslinked. The atomic group having a sugar phosphate skeleton at anucleotide residue is not particularly limited, and examples thereofinclude atomic groups having sugar phosphate skeletons at known naturalor artificial nucleotide residues. Examples of the atomic group having asugar phosphate skeleton at a natural nucleotide residue include anatomic group having a ribose phosphate skeleton at a ribonucleotideresidue and an atomic group having a deoxyribose phosphate skeleton on adeoxyribonucleotide. The atomic group having a sugar phosphate skeletonat an artificial nucleoside residue can be, for example, an atomic grouphaving a bicyclic sugar phosphate skeleton at an artificial nucleosideresidue, and specific examples thereof can be an atomic group having aribose phosphate skeleton where an oxygen atom at 2′-position and acarbon atom at 4′ position of 2′-O,4′-C-Ethylene-bridged Nucleic Acids(ENA) or Locked Nucleic Acid (LNA) is crosslinked.

In the chemical formula (1), an atomic group having a sugar skeleton ata nucleoside residue or an atomic group having a sugar phosphateskeleton at a nucleotide residue is represented by preferably thefollowing chemical formula (2).

In the chemical formula (2), R² is a hydrogen atom, a protecting group,or a group represented by the following chemical formula (3), R³ is ahydrogen atom, a protecting group, or a phosphoramidite group, R⁴ is ahydrogen atom, a fluorine atom, a hydroxyl group, an amino group, or amercapto group, and

in the chemical formula (3), Y is an oxygen atom or a sulfur atom, Z isa hydroxyl group or an imidazole group, and m is an integer of 1 to 10.

In the chemical formula (2), R² is a hydrogen atom, a protecting group,or a group represented by the following chemical formula (3). Theprotecting group is not particularly limited and can be, for example, aprotecting group of a known hydroxyl group used in nucleic acidsynthesis methods, and as a specific example, the protecting group canbe an DMTr group (4,4′-dimethoxy(triphenylmethyl) group). When R² is agroup represented by the chemical formula (3), the nucleoside derivativeof the present invention can also be referred to as a nucleotidederivative, for example.

In the chemical formula (2), R³ is a hydrogen atom, a protecting group,or a phosphoramidite group. The protecting group is not particularlylimited, and, the description of R² can be incorporated in thedescription of the protecting group by reference, for example. Thephosphoramidite group is represented by the chemical formula (5). WhenR³ is a phosphoramidite group, the nucleoside derivative of the presentinvention can also be referred to as a phosphoramidite compound of thenucleoside derivative, for example. When R² is a group represented bythe chemical formula (3), and R³ is a phosphoramidite group, thenucleoside derivative of the present invention can also be referred toas, for example, a phosphoramidite compound of the nucleotidederivative.

In the chemical formula (2), R⁴ is a hydrogen atom, a fluorine atom, ahydroxyl group, an amino group, or a mercapto group and is preferably ahydrogen atom or a hydroxyl group. When R⁴ is a hydrogen atom, thenucleoside derivative of the present invention has a deoxyriboseskeleton as a sugar skeleton and can be used for, for example, synthesisof DNAs. When R⁴ is a hydroxyl group, the nucleoside derivative of thepresent invention has a ribose skeleton as a sugar skeleton and can beused for, for example, synthesis of RNAs.

In the chemical formula (3), Y is an oxygen atom or a sulfur atom. WhenY is an oxygen atom, polynucleotide including, as a building block, thenucleoside derivative of the present invention can also be referred toas polynucleotide having a phosphodiester bond. When Y is a sulfur atom,polynucleotide including, as a building block, the nucleoside derivativeof the present invention can also be referred to as polynucleotidehaving a phosphorothioate bond.

In the chemical formula (3), Z is a hydroxyl group or an imidazolegroup. In the imidazole group, imidazole is bound to a phosphate atomvia a nitrogen atom at the 1-position, for example.

In the chemical formula (3), m is an integer of 1 to 10, preferably 1 to3, 1 to 2, or 1.

The nucleoside derivative of the present invention is represented bypreferably the following chemical formula (4), (6), (7), or (8). Therespective nucleoside derivatives represented by the following chemicalformulae (4), (6), (7), and (8) are also referred to as MK4, MK1, MK2,and MK3.

The nucleoside derivative or a salt thereof of the present invention maybe a stereoisomer such as enantiomers, tautomers, geometric isomers,conformers, and optical isomers thereof, and salts thereof.Specifically, in the chemical formula (2) and chemical formulaedescribed below, the sugar skeleton is D body, but the nucleosidederivative of the present invention is not limited thereto, and thesugar skeleton may be L body.

The salt of the nucleoside derivative of the present invention may be anacid addition salt or a base addition salt. Further, acid which formsthe acid addition salt may be an inorganic acid or an organic acid, andbase which forms the base addition salt may be an inorganic base or anorganic base. The inorganic acid is not particularly limited, andexamples thereof include sulfuric acid, phosphoric acid, hydrofluoricacid, hydrochloric acid, hydrobromic acid, hydroiodic acid, hypofluorousacid, hypochlorous acid, hypobromous acid, hypoiodous acid, fluorousacid, chlorous acid, bromous acid, iodous acid, fluorine acid, chloricacid, bromic acid, iodine acid, perfluoric acid, perchloric acid,perbromic acid, and periodic acid. The organic acid is not particularlylimited, and examples thereof include p-toluenesulfonic acid,methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, and acetic acid. Theinorganic base is not particularly limited, and examples thereof includeammonium hydroxides, alkali metal hydroxides, alkaline earth metalhydroxides, carbonates, and bicarbonates. More specific examples thereofinclude sodium hydroxide, potassium hydroxide, potassium carbonate,sodium carbonate, sodium bicarbonate, potassium hydrogen carbonate,calcium hydroxide, and calcium carbonate. The organic base is notparticularly limited, and examples thereof include ethanolamine,triethylamine, and tris(hydroxymethyl)aminomethane.

The method for producing the nucleoside derivative of the presentinvention is not particularly limited, and the nucleoside derivative ofthe present invention can be produced by combining known synthesismethods. As a specific example, the nucleoside derivative of the presentinvention can be synthesized by, for example, an amidation reactionbetween a nucleoside derivative into which an acrylic acid structure isintroduced and an adenine in which a substituent having an amino groupat a terminal thereof is substituted with a hydrogen atom of an aminogroup, as in the synthetic method of the example described below.

(Polynucleotide Synthesis Reagent)

The polynucleotide synthesis reagent (hereinafter also referred to as“synthesis reagent”) of the present invention contains a nucleotidederivative or a salt thereof including the nucleoside derivative or asalt thereof of the present invention, as mentioned above. The synthesisreagent of the present invention is characterized by containing thenucleoside derivative of the present invention, and other compositionand conditions are not particularly limited. The description of thenucleoside derivative or a salt thereof of the present invention can beincorporated in the description of the synthesis reagent of the presentinvention by reference, for example. The synthesis reagent of thepresent invention can be described with reference to the description ofthe polynucleotide of the present invention, for example.

In the synthesis reagent of the present invention, the nucleosidederivative preferably contains at least one of the phosphoramiditecompound or the nucleotide derivative, for example.

The synthesis reagent of the present invention may further containanother reagent for use in synthesis of polynucleotide, for example.

(Method for Producing Polynucleotide)

The method for producing a polynucleotide of the present inventionincludes, as mentioned above, the step of synthesizing a polynucleotideusing a nucleotide derivative or a salt thereof including the nucleosidederivative or a salt thereof of the present invention. The method forproducing a polynucleotide of the present invention is characterized byusing a nucleotide derivative or a salt thereof including the nucleosidederivative or a salt thereof of the present invention in the synthesisstep, and other steps and conditions are not particularly limited. Thedescriptions of the nucleoside derivative or a salt thereof and thesynthesis reagent of the present invention can be incorporated in themethod for producing a polynucleotide of the present invention byreference, for example. By the method for producing a polynucleotide ofthe present invention, the polynucleotide of the present invention to bedescribed below can be produced, for example.

In the method for producing a polynucleotide of the present invention,the synthesis reagent of the present invention may be used as thenucleotide derivative or a salt thereof including the nucleosidederivative or a salt thereof of the present invention.

In the synthesis step, the method for synthesizing the polynucleotide isnot particularly limited, and the polynucleotide can be synthesized by aknown polynucleotide synthesis method.

When the phosphoramidite compound is used as the nucleotide derivativeor a salt thereof, the polynucleotide can be synthesized by aphosphoramidite method in the synthesis step.

The method for producing the polynucleotide of the present invention mayfurther include a step of purifying the polynucleotide obtained in thesynthesis step, for example. The purification method in the purificationstep is not particularly limited, and the polynucleotide can be purifiedby a known purification method such as column chromatography.

(Polynucleotide)

As mentioned above, the polynucleotide of the present inventionincludes, as a building block, a nucleotide derivative or a salt thereofincluding the nucleoside derivative or a salt thereof of the presentinvention. The method for producing a polynucleotide of the presentinvention is characterized by using a nucleotide derivative or a saltthereof including the nucleoside derivative or a salt thereof of thepresent invention in the synthesis step, and other steps and conditionsare not particularly limited. The descriptions of the nucleosidederivative or a salt thereof, the polynucleotide synthesis reagent, andthe method for producing a polynucleotide of the present invention canbe incorporated in the description of the polynucleotide of the presentinvention by reference, for example. With the polynucleotide of thepresent invention, a binding nucleic acid molecule that binds to atarget can be produced, for example, as mentioned below. In thepolynucleotide of the present invention, the building block means, forexample, a part of the polynucleotide.

The polynucleotide of the present invention has a structure representedby the following chemical formula (9), for example. The description ofeach substituent can be incorporated in the description of eachsubstituent in the chemical formula (9) by reference, for example.

The polynucleotide of the present invention can be, for example, abinding nucleic acid molecule that binds to a target. The target is notparticularly limited and can be any target, and as a specific example,the target can be a biomolecule. Examples of the biomolecule includesecretory immunoglobulin A (sIgA), an amylase (e.g., α-amylase),chromogranin A, β-defensin (Defensin) 2, β-defensin 4A, kallikrein,C-reactive proteins (CRPs), calprotectin, Statherins, cortisol,melatonin, lysozyme, lactate dehydrogenase (LDH)5, and interleukin(IL)-6. The binding nucleic acid molecule can be produced by the methodfor producing a binding nucleic acid molecule of the present inventionto be described below.

The polynucleotide of the present invention may further include, forexample, other nucleotide in addition to the nucleotide derivative.Examples of the nucleotide include deoxyribonucleotide andribonucleotide. Examples of the polynucleotide of the present inventioninclude DNA consisting of deoxyribonucleotide only, DNA/RNA includingdeoxyribonucleotide and ribonucleotide, and RNA consisting ofribonucleotide only. Other nucleotide may be, for example, a modifiednucleotide.

Examples of the modified nucleotide include modified deoxyribonucleotideand modified ribonucleotide. The modified nucleotide can be, forexample, a nucleotide with a modified sugar. Examples of the sugarinclude deoxyribose and ribose. The modified site in the nucleotide isnot particularly limited, and may be, for example, the 2′-position orthe 4′-position of the sugar. Examples of the modification includemethylation, fluorination, amination, and thiation. The modifiednucleotide can be, for example, a modified nucleotide with a pyrimidinebase (pyrimidine nucleus) as a base or a modified nucleotide with apurine base (purine nucleus) as a base and is preferably the former.Hereinafter, a nucleotide with a pyrimidine base is referred to aspyrimidine nucleotide, the pyrimidine nucleotide modified is referred toas modified pyrimidine nucleotide, a nucleotide with a purine base isreferred to as purine nucleotide, and the purine nucleotide modified isreferred to as modified purine nucleotide. Examples of the pyrimidinenucleotide include an uracil nucleotide with uracil, cytosine nucleotidewith cytosine, and thymine nucleotide with thymine. When the base in themodified nucleotide is a pyrimidine base, it is preferable that the2′-position and/or the 4′-position of the sugar is modified, forexample. Specific examples of the modified nucleotide include modifiednucleotides with the 2′-position of the ribose being modified, such as a2′-methylated-uracil nucleotide, 2′-methylated-cytosine nucleotide,2′-fluorinated-uracil nucleotide, 2′-fluorinated-cytosine nucleotide,2′-aminated-uracil nucleotide, 2′-aminated-cytosine nucleotide,2′-thiated-uracil nucleotide, and 2′-thiated-cytosine nucleotide.

The base in the other nucleotide may be, for example, a natural base(non-artificial base) such as adenine (A), cytosine (C), guanine (G),thymine (T), and uracil (U), or a non-natural base (artificial base).Examples of the artificial base include modified bases and alteredbases. The artificial base preferably has the same function as thenatural base (A, C, G, T, or U). Example of the artificial base havingthe same function as the natural base include artificial bases capableof binding to cytosine (C) instead of guanine (G), capable of binding toguanine (G) instead of cytosine (C), capable of binding to thymine (T)or uracil (U) instead of adenine (A), capable of binding to adenine (A)instead of thymine (T), and capable of binding to adenine (A) instead ofuracil (U). The modified base is not particularly limited, and may be,for example, a methylated base, a fluorinated base, aminated base, andthiated base. Specific examples of the modified base include2′-methyluracil, 2′-methylcytosine, 2′-fluorouracil, 2′-fluorocytosine,2′-aminouracil, 2′-aminocytosine, 2′-thiouracil, and 2′-thiocytosine. Inthe present invention, for example, the bases represented by A, G, C, T,and U include the meaning of, in addition to the natural bases, theartificial bases having the same functions as the natural bases.

The polynucleotide of the present invention may further include, forexample, an artificial nucleic acid monomer in addition to thenucleotide derivative. Examples of the artificial nucleic acid monomerinclude peptide nucleic acids (PNAs), LNAs, and ENAs. The base in themonomer residue is the same as described above, for example.

The length of the polynucleotide of the present invention is notparticularly limited, and the lower limit thereof is, for example,10-mer, 20-mer, or 25-mer, the upper limit thereof is, for example,150-mer, 100-mer, or 70-mer, and the range thereof is, for example, 10-to 150-mer, 20- to 100-mer, or 25- to 70-mer.

The polynucleotide of the present invention may further include anadditional sequence, for example. Preferably, the additional sequence isbound to at least one of the 5′ end or the 3′ end, more preferably tothe 3′ end of the polynucleotide, for example. The additional sequenceis not particularly limited, and the length thereof is also notparticularly limited.

The polynucleotide of the present invention may further include alabeling substance, for example. Preferably, the labeling substance isbound to at least one of the 5′ end or the 3′ end, more preferably tothe 5′ end of the polynucleotide, for example. The labeling substance isnot particularly limited, and examples thereof include fluorescentsubstances, dyes, isotopes, and enzymes. Examples of the fluorescentsubstances include pyrenes, TAMRA, fluorescein, Cy®3 dyes, Cy®5 dyes,FAM dyes, rhodamine dyes, Texas Red dyes, fluorophores such as JOE, MAX,HEX, and TYE, and examples of the dyes include Alexa dyes such asAlexa®488 and Alexa®647.

The labeling substance may, for example, be linked directly to thenucleic acid molecule or linked indirectly via the additional sequence.

The polynucleotide of the present invention can be used in the statewhere it is immobilized on a carrier, for example. It is preferable toimmobilize either the 5′ end or the 3′ end, more preferably the 3′ endof the polynucleotide of the present invention, for example. When thepolynucleotide of the present invention is immobilized, thepolynucleotide may be immobilized either directly or indirectly on thecarrier, for example. In the latter case, it is preferable to immobilizethe nucleic acid molecule via the additional sequence, for example.

(Method for Producing Binding Nucleic Acid Molecule)

The method for producing a binding nucleic acid molecule of the presentinvention includes, as mentioned above, the steps of: causing acandidate polynucleotide and a target to come into contact with eachother; and selecting the candidate polynucleotide bound to the target asa binding nucleic acid molecule that binds to the target, and thecandidate polynucleotide is the polynucleotide of the present invention.The method for producing a binding nucleic acid molecule of the presentinvention is characterized in that the candidate polynucleotide is thepolynucleotide of the present invention, for example, and other steps,conditions, etc. are not particularly limited. The descriptions of thenucleoside derivative or a salt thereof, the synthesis reagent, themethod for producing polynucleotide, and the polynucleotide can beincorporated in the method for producing a binding nucleic acid of thepresent invention by reference, for example. In the method for producinga binding nucleic acid molecule of the present invention, the candidatepolynucleotide includes, as a building block, a nucleotide derivative ora salt thereof including the nucleoside derivative or a salt thereof ofthe present invention. Thus, for example, a binding nucleic acidmolecule that exhibits excellent binding ability to a target can beproduced by the method for producing a binding nucleic acid molecule ofthe present invention.

As to the binding nucleic acid molecule of the present invention, thecontact step and the selection step can be performed by the SELEXmethod, for example.

The number of candidate polynucleotides in the contact step is notparticularly limited, and the number of candidate polynucleotides in thecontact step is, for example, 4²⁰ to 4¹²⁰ types (about 10¹² to 10⁷²) and4³⁰ to 4⁶⁰ types (about 10¹⁸ to 10³⁶).

In the contact step, a candidate polynucleotide and a target are causedto come into contact with each other. Then, by the contact, thecandidate polynucleotide and the target are reacted to form a complexbetween the candidate polynucleotide and the target. The target to beused in the contact step may be, for example, the target itself or adecomposition product thereof. The conditions under which the candidatepolynucleotide and the target are bound are not particularly limited,and for example, the binding can be performed by incubating the both ina solvent for a certain period of time. The solvent is not particularlylimited, and for example, a solvent in which the binding of the both isretained is preferable, and specific examples thereof include variousbuffer solutions.

Next, in the selecting step, a candidate polynucleotide bound to thetarget is selected as a binding nucleic acid molecule that binds to thetarget. Specifically, a candidate polynucleotide that forms a complexwith the target is collected as the binding nucleic acid molecule. Amixture of the candidate polynucleotide and the target after the contactstep contains, in addition to the complex, a candidate polynucleotidethat is not involved in formation of the complex, for example. Thus, itis preferable that the complex and unreacted candidate polynucleotideare separated from each other from the mixture, for example. Theseparation method is not particularly limited and can be, for example, amethod utilizing a difference in adsorbability between the target andthe candidate polynucleotide or a difference in molecular weight betweenthe complex and the candidate polynucleotide.

In addition to this method, the separation method can be, for example, amethod using a target immobilized on a carrier in formation of thecomplex. That is, the target is immobilized on a carrier in advance tocontact between the carrier and the candidate polynucleotide, therebyforming a complex the immobilized target and the candidatepolynucleotide. An unreacted candidate polynucleotide that does not bindto the immobilized target is then removed, and the complex between thetarget and the candidate polynucleotide is dissociated from the carrier.The method for immobilizing the target on a carrier is not particularlylimited and can be carried out by a known method. The carrier is notparticularly limited, and, a known carrier can be used.

In the above-described manner, the binding nucleic acid molecule thatbinds to a target can be produced.

The method for producing a binding nucleic acid molecule of the presentinvention may further include, for example, the step of determining abase sequence of the selected binding nucleic acid molecule. The methodfor determining the base sequence is not particularly limited, and thebase sequence can be determined by a known base sequence determinationmethod.

In the method for producing a binding nucleic acid molecule of thepresent invention, for example, one set of the contact step and theselection step may be performed for two or more cycles in total, and aspecific example thereof is 3 to 15 cycles.

(α-amylase-Binding Nucleic Acid Molecule)

The α-amylase-binding nucleic acid molecule (hereinafter also referredto as “α-amylase nucleic acid molecule”) of the present inventionincludes the following polynucleotide (a):

(a) a polynucleotide (a1):(a1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:1 and 11 to 16.

The α-amylase nucleic acid molecule of the present invention can bind toα-amylase, as mentioned above. The α-amylase is not particularlylimited, and the α-amylase may be derived from a human or a non-humananimal, for example. Examples of the non-human animal include mice,rats, monkeys, rabbits, dogs, cats, horses, cows, and pigs. Amino acidsequence information on human α-amylase is registered under AccessionNo. P04745 in UniProt (http://www.uniprot.org/), for example.

In the present invention, the expression “binds to α-amylase” (andgrammatical variations thereof) is also referred to as “has bindingability to α-amylase” or “has binding activity to α-amylase”, forexample. The binding between the nucleic acid molecule of the presentinvention and the α-amylase can be determined by surface plasmonresonance (SPR) analysis or the like, for example. The analysis can beperformed using ProteON (trade name, BioRad), for example. Since theα-amylase nucleic acid molecule of the present invention binds toα-amylase, it can be used for detection of the α-amylase, for example.

As mentioned above, the α-amylase nucleic acid molecule of the presentinvention comprises the following polynucleotide (a):

(a) a polynucleotide (a1):(a1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:1 and 11 to 16.

α-amylase-binding nucleic acid molecule 1 (SEQ ID NO: 1)5′-GGTTTGGACGCAATCTCCCTAATCTAGTGACGAAAATGTACGAGGGGGTCATTTGAAACTACAATGGGCGGGCTTATC-3′α-amylase-binding nucleic acid molecule 2 (SEQ ID NO: 11)5′-GGTTTGGACGCAATCTCCCTAATCTAGTGACGAAAATGTACGAG GGGGTCATTTGAAACTA-3′α-amylase-binding nucleic acid molecule 3 (SEQ ID NO: 12)5′-GCAATCTCCCTAATCTAGTGACGAAAATGTACGAGGGGGTCATT TGAAACTA-3′α-amylase-binding nucleic acid molecule 4 (SEQ ID NO: 13)55′-GGTTTGGACGCAATCTCCCTAATCAGACTATTATTTCAAGTACGTGGGGGTCTTGAAACTACAATGGGCGGGCTTATC-3′α-amylase-binding nucleic acid molecule 5 (SEQ ID NO: 14)5′-GGTTTGGACGCAATCTCCCTAATCTAAAGTTTCTAAACGATGTGGCGGCATTCAGAAACTACAATGGGCGGGCTTATC-3′α-amylase-binding nucleic acid molecule 6 (SEQ ID NO: 15)5′-GGTTTGGACGCAATCTCCCTAATCTAAAGTTTCTAAACGATGTG GCGGCATTCAGAAACT-3′α-amylase-binding nucleic acid molecule 7 (SEQ ID NO: 16)5′-GCAATCTCCCTAATCTAAAGTTTCTAAACGATGTGGCGGCATTC AGAAACT-3′′

The polynucleotide (a) above also includes, for example, the meaning ofthe polynucleotide of (a2), (a3), or (a4) below:

(a2) a polynucleotide consisting of a base sequence obtained bydeletion, substitution, insertion, and/or addition of one or more basesin any of the base sequences of the polynucleotide (a1) and binds to theα-amylase;(a3) a polynucleotide consisting of a base sequence having at least 80%sequence identity to any of the base sequences of the polynucleotide(a1) and binds to the α-amylase; and(a4) a polynucleotide consisting of a base sequence complementary to apolynucleotide hybridizing to any of the base sequences of thepolynucleotide (a1) under stringent conditions and binds to theα-amylase.

Regarding the polynucleotide (a2), the term “one or more” is not limitedas long as, for example, it is in the range where the polynucleotide(a2) binds to α-amylase. The number of the “one or more” bases is, forexample, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 or 2, or 1. In thepresent invention, the numerical range regarding the number of bases,sequences, or the like discloses, for example, all the positive integersfalling within that range. That is, for example, the description “one tofive bases” discloses all of “one, two, three, four, and five bases”(the same applies hereinafter).

Regarding the polynucleotide (a3), the “sequence identity” is notlimited as long as, for example, it is in the range where thepolynucleotide (a3) binds to α-amylase. The sequence identity is, forexample, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%. The sequenceidentity can be calculated with analysis software such as BLAST or FASTAusing default parameters, for example (the same applies hereinafter).

Regarding the polynucleotide (a4), the “polynucleotide hybridizing to”may be, for example, a polynucleotide that is perfectly or partiallycomplementary to the polynucleotide (a1) and binds to the α-amylase. Thehybridization can be detected by various types of hybridization assay,for example. The hybridization assay is not particularly limited, andfor example, a method described in “Molecular Cloning: A LaboratoryManual 2^(nd) Ed.” edited by Sambrook et al. (Cold Spring HarborLaboratory Press (1989)) or the like can be employed.

Regarding the polynucleotide (a4), the “stringent conditions” may be anyof low stringency conditions, medium stringency conditions, and highstringency conditions, for example. The “low stringency conditions” are,for example, conditions where 5×SSC, 5× Denhardt's solution, 0.5% SDS,and 50% formamide are used at 32° C. The “medium stringency conditions”are, for example, conditions where 5×SSC, 5× Denhardt's solution, 0.5%SDS, and 50% formamide are used at 42° C. The “high stringencyconditions” are, for example, conditions where 5×SSC, 5 x Denhardt'ssolution, 0.5% SDS, and 50% formamide, are used at 50° C. Those skilledin the art can set the degree of stringency by, for example, setting theconditions such as the temperature, the salt concentration, theconcentration and length of a probe, the ionic strength, the time, etc.as appropriate. As the “stringent conditions”, it is also possible toemploy conditions described in the above-described “Molecular Cloning: ALaboratory Manual 2^(nd) Ed.” edited by Sambrook et al. (Cold SpringHarbor Laboratory Press (1989)) or the like, for example.

In the α-amylase nucleic acid molecule of the present invention, thebuilding blocks of the polynucleotide are, for example, nucleotideresidues, examples of which include deoxyribonucleotide residues andribonucleotide residues. The polynucleotide is, for example, a DNAconsisting of deoxyribonucleotide residues or a DNA including adeoxyribonucleotide residue(s) and a ribonucleotide residue(s), and thepolynucleotide may further include a non-nucleotide residue(s), asmentioned below. The α-amylase-binding nucleic acid molecule of thepresent invention may be also referred to as “α-amylase aptamer”hereinafter, for example.

The α-amylase nucleic acid molecule of the present invention may consistof any of the above-described polynucleotides, or may include any of theabove-described polynucleotides, for example. In the latter case, theα-amylase nucleic acid molecule of the present invention may include,for example, two or more polynucleotides selected from theabove-described polynucleotides, as mentioned below. The two or morepolynucleotides may be the polynucleotides with the same sequence ordifferent sequences. Also, in the latter case, the α-amylase nucleicacid molecule of the present invention may further include a linker(s)and/or an additional sequence(s), for example. The linker is a sequencepresent between polynucleotides, for example. The additional sequence isa sequence added to an end, for example.

When the α-amylase nucleic acid molecule of the present inventionincludes, for example, a plurality of polynucleotides selected from theabove-described polynucleotides, it is preferable that the plurality ofpolynucleotide sequences are linked to each other to form asingle-stranded polynucleotide. The plurality of polynucleotidesequences may be linked to each other directly, or may be linked to eachother indirectly with a linker, for example. It is preferable that thepolynucleotide sequences are linked to each other directly or indirectlyat their ends. When the α-amylase nucleic acid molecule of the presentinvention includes the plurality of polynucleotide sequences, the numberof the sequences is not particularly limited, and is, for example, 2 ormore, 2 to 20, 2 to 10, or 2 or 3.

The length of the linker is not particularly limited, and is, forexample, 1- to 200-mer, 1-to 20-mer, 3- to 12-mer, or 5- to 9-mer. Thebuilding blocks of the linker are, for example, nucleotide residues,examples of which include deoxyribonucleotide residues andribonucleotide residues. The linker is not particularly limited, andexamples thereof include polynucleotides such as a DNA consisting ofdeoxyribonucleotide residues and a DNA including a ribonucleotideresidue(s). Specific examples of the linker include polydeoxythymine(poly[dT]), polydeoxyadenine (poly[dA]), and poly(dA-dT) having arepetitive sequence composed of A and T. Preferably, the linker ispoly(dT) or poly(dA-dT).

In the α-amylase nucleic acid molecule of the present invention, thepolynucleotide is preferably a single-stranded polynucleotide. It ispreferable that the single-stranded polynucleotide can form a stemstructure and a loop structure by self-annealing, for example. It ispreferable that the polynucleotide can form a stem-loop structure, aninternal loop structure, and/or a bulge structure, for example.

The α-amylase nucleic acid molecule of the present invention may be adouble strand, for example. When the α-amylase nucleic acid molecule isa double strand, for example, one of single-stranded polynucleotidesincludes the polynucleotide (a), and the other single-strandedpolynucleotide is not limited. The other single-stranded polynucleotidemay be, for example, a polynucleotide including a base sequencecomplementary to the polynucleotide (a). When the α-amylase nucleic acidmolecule of the present invention is a double strand, it is preferableto dissociate the double strand to single-stranded polynucleotides bydenaturation or the like before use, for example. Also, it is preferablethat the dissociated single-stranded polynucleotide including thepolynucleotide (a) is forming a stem structure and a loop structure asmentioned above, for example.

In the present invention, the expression “can form a stem structure anda loop structure” encompasses that, for example, a stem structure and aloop structure are formed actually, and also, even if a stem structureand a loop structure are not formed, they can be formed depending onconditions. The expression “can form a stem structure and a loopstructure (and grammatical variations thereof)” encompasses, forexample, both the cases where the formation thereof has been confirmedthrough an experiment and where the formation thereof is predictedthrough simulation using a computer or the like.

The building blocks of the α-amylase nucleic acid molecule of thepresent invention are, for example, nucleotide residues. Examples of thenucleotide residues include deoxyribonucleotide residues andribonucleotide residues. The α-amylase nucleic acid molecule of thepresent invention may be, for example, a DNA consisting ofdeoxyribonucleotide residues only or a DNA including one or moreribonucleotide residues. In the latter case, “one or more” is notparticularly limited. For example, the number of the ribonucleotideresidues in the polynucleotide is, for example, 1 to 91, 1 to 30, 1 to15, 1 to 7, 1 to 3, or 1 or 2.

The polynucleotide may include, as a base in a nucleotide residue, anatural base or a modified base. The natural base (non-artificial base)is not particularly limited, and may be, for example, a purine base witha purine skeleton or a pyrimidine base with a pyrimidine skeleton. Thepurine base is not particularly limited, and examples thereof includeadenine (A) and guanine (G). The pyrimidine base is not particularlylimited, and examples thereof include cytosine (C), thymine (T), anduracil (U). Among them, cytosine (C) and thymine (T) are preferable.

When the polynucleotide includes the modified base(s), the site and thenumber of the modified bases are not particularly limited. When thepolynucleotide (a) has the modified base(s), some or all of theunderlined adenines in the polynucleotide consisting of any of basesequences of SEQ ID NOs: 1 and 11 to 16 are modified bases, for example.When the underlined adenine is the modified base, the modified base is amodified purine base, which is a purine base modified with a modifyinggroup.

The modified base is a base modified with a modifying group, forexample. The base to be modified with the modifying group (also referredto simply as the “base to be modified” hereinafter) is the natural base,for example. The natural base is not particularly limited, and may be,for example, a purine base or a pyrimidine base. The modified base isnot particularly limited, and may be, for example, a modified adenine, amodified guanine, a modified cytosine, a modified thymine, or a modifieduracil.

In the modified base, the base to be modified may be modified with themodifying group either directly or indirectly, for example. In thelatter case, the base to be modified may be modified with the modifyinggroup via a linker, for example. The linker is not particularly limited.

In the base to be modified, a site to be modified with the modifyinggroup is not particularly limited. When the base is a purine base, themodified site in the purine base may be, for example, the 7-position orthe 8-position, preferably the 7-position of the purine skeleton. Whenthe modified site in the purine base is the 7-position of the purineskeleton, the nitrogen atom at the 7-position is preferably substitutedwith a carbon atom. When the base is a pyrimidine base, the modifiedsite in the pyrimidine base may be, for example, the 5-position or the6-position, preferably the 5-position of the pyrimidine skeleton.Thymine has a methyl group bound to carbon at the 5-position. Thus, whenthe 5-position of the pyrimidine base is modified, for example, themodifying group may be bound to the carbon at the 5-position eitherdirectly or indirectly, or the modifying group may be bound to carbon inthe methyl group bound to the carbon at the 5-position either directlyor indirectly. When the pyrimidine skeleton has “═O” bound to carbon atthe 4-position and a group that is not “—CH₃” or “—H” bound to carbon atthe 5-position, the modified base can be referred to as a modifieduracil or a modified thymine.

When the modified base is a modified purine base, the modifying group ispreferably an adenine residue. That is, the modified purine base is abase modified with an adenine residue, for example. In the base to bemodified, a site to be modified with the adenine residue (binding siteof the adenine residue to the base to be modified) is not particularlylimited, and can be, for example, an amino group that binds to carbon atthe 6-position of the adenine residue. The base to be modified with theadenine residue is not particularly limited, and is preferably purinebase, for example, and it is preferable that atom at the 7-position ofthe purine base is modified with the adenine residue. When the modifiedbase is a modified thymine base, the modifying group is preferably anadenine residue or a guanine base. That is, the modified base is, forexample, a base modified with an adenine residue or a guanine residue.In the base to be modified, a site to be modified with the adenineresidue is not particularly limited, and can be, for example, an aminogroup that binds to carbon at the 6-position of the adenine residue. Inthe base to be modified, a site to be modified with the guanine residueis not particularly limited, and can be, for example, an amino groupthat binds to carbon at the 2-position of the guanine residue. The baseto be modified with the adenine residue or the guanine residue is notparticularly limited, and is preferably a thymine, for example, and itis preferable that carbon in a methyl group bound to the carbon at the5-position of the thymine is modified with the adenine residue or theguanine residue.

When the modifying group is the adenine residue or the guanine residue,it is preferable that, for example, the base to be modified is modifiedwith the modifying group via the linker, as shown below.

[nucleotide residue]-[linker]-[adenine residue][nucleotide residue]-[linker]-[guanine residue]

The linker is not particularly limited, and can be represented by, forexample, each formula present between the nucleotide residue and theadenine residue/guanine residue, as shown below. It is to be noted,however, that the linker is not limited thereto. In each formula, thenumerical value “n” in (CH₂)_(n) is 1 to 10, 2 to 10, or 2, for example.

[nucleotide residue]═C—C(═O)—NH—(CH₂)_(n)-[adenine residue][nucleotide residue]═C—C(═O)—NH—(CH₂)_(n)-[guanine residue][nucleotide residue]C═C—C(═O)—NH—(CH₂)_(n)-[adenine residue][nucleotide residue]═C—C(═O)—NH—CH₂—CH₂-[adenine residue][nucleotide residue]═C—C(═O)—NH—CH₂—CH₂-[guanine residue][nucleotide residue]—C═C—C(═O)—NH—CH₂—CH₂-[adenine residue]

In each formula, one ends of the linker [═C] and [—C] form a double bondand a single bond with carbon of the base to be modified in thenucleotide residue, respectively, for example, and the other end of thelinker [CH₂—] is bound to amine (—NH) in the guanine residue or theadenine residue, for example.

Specific examples of an adenosine nucleotide residue modified with theadenine residue in the polynucleotide include a residue represented bythe following chemical formula (10) (also referred to as “nucleotideresidue of MK4” hereinafter). It is to be noted, however, that thepresent invention is not limited thereto.

In the polynucleotide consisting of any of base sequences of SEQ ID NOs:1 and 11 to 16, it is more preferable that the underlined adenine is anucleotide residue of the MK4.

When the α-amylase nucleic acid molecule of the present inventionincludes the nucleotide residues of the MK4, the polynucleotide can besynthesized using, as a monomer molecule, a nucleotide triphosphaterepresented by the following chemical formula (4) (hereinafter alsoreferred to as “MK4 monomer” hereinafter), for example. In the synthesisof the polynucleotide, for example, the monomer molecule binds toanother nucleotide triphosphate via a phosphodiester bond. A method forproducing the MK4 monomer is described below.

Other examples of the modifying group include a methyl group, a fluorogroup, an amino group, a thio group, a benzylaminocarbonyl group, atryptaminocarbonyl group, and an isobutylaminocarbonyl group.

Specific examples of the modified adenine include 7′-deazaadenine.Specific examples of the modified guanine include 7′-deazaguanine.Specific examples of the modified cytosine include 5′-methylcytosine(5-Me-dC). Specific examples of the modified thymine include5′-benzylaminocarbonyl thymine, 5′-tryptaminocarbonyl thymine, and5′-isobutylaminocarbonyl thymine. Specific examples of the modifieduracil include 5′-benzylaminocarbonyl uracil (BndU),5′-tryptaminocarbonyl uracil (TrpdU), and 5′-isobutylaminocarbonyluracil. The modified uracils given above as examples can be alsoreferred to as modified thymines.

The polynucleotide may include only one type or two or more types of themodified bases, for example.

The α-amylase nucleic acid molecule of the present invention may includea modified nucleotide, for example. The modified nucleotide may be anucleotide having the above-described modified base, a nucleotide havinga modified sugar obtained through modification of a sugar residue, or anucleotide having the modified base and the modified sugar.

The sugar residue is not particularly limited, and may be a deoxyriboseresidue or a ribose residue, for example. The modified site in the sugarresidue is not particularly limited, and may be, for example, the2′-position or the 4′-position of the sugar residue. Either one or bothof the 2′-position and the 4′-position may be modified. Examples of amodifying group in the modified sugar include a methyl group, a fluorogroup, an amino group, a thio group.

When the base in the modified nucleotide residue is a pyrimidine base,it is preferable that the 2′-position and/or the 4′-position of thesugar residue is modified, for example. Specific examples of themodified nucleotide residue include modified nucleotide residues withthe 2′-position of the deoxyribose residue or ribose residue beingmodified, such as a 2′-methylated-uracil nucleotide residue,2′-methylated-cytosine nucleotide residue, 2′-fluorinated-uracilnucleotide residue, 2′-fluorinated-cytosine nucleotide residue,2′-aminated-uracil nucleotide residue, 2′-aminated-cytosine nucleotideresidue, 2′-thiated-uracil nucleotide residue, and 2′-thiated-cytosinenucleotide residue.

The number of the modified nucleotides is not particularly limited. Forexample, the number of the modified nucleotides in the polynucleotideis, for example, 1 to 100, 1 to 90, 1 to 80, or 1 to 70. Also, thenumber of the modified nucleotides in the full-length nucleic acidmolecule including the polynucleotide is not particularly limited, andis, for example, 1 to 91, 1 to 78, or in the numerical ranges givenabove as examples of the number of the modified nucleotides in thepolynucleotide.

The α-amylase nucleic acid molecule of the present invention mayinclude, for example, one or more artificial nucleic acid monomerresidues. The term “one or more” is not particularly limited, and maybe, for example, 1 to 100, 1 to 50, 1 to 30, or 1 to 10 in thepolynucleotide, for example. Examples of the artificial nucleic acidmonomer residue include peptide nucleic acids (PNAs), locked nucleicacids (LNAs), and 2′-O,4′-C-ethylenebridged nucleic acids (ENAs). Thenucleic acid in the monomer residue is the same as described above, forexample.

It is preferable that the α-amylase nucleic acid molecule of the presentinvention is resistant to nuclease, for example. In order to allow theα-amylase nucleic acid molecule of the present invention to havenuclease resistance, it is preferable that the nucleic acid molecule ofthe present invention includes the modified nucleotide residue(s) and/orthe artificial nucleic acid monomer residue(s), for example. Also, inorder to allow the α-amylase nucleic acid molecule of the presentinvention to have nuclease resistance, the nucleic acid molecule of thepresent invention may have polyethylene glycol (PEG) of several tens ofkDa, deoxythymidine, or the like bound to, e.g., the 5′ end or the 3′end thereof.

The α-amylase nucleic acid molecule of the present invention may furtherinclude an additional sequence, for example. Preferably, the additionalsequence is bound to at least one of the 5′ end and the 3′ end, morepreferably to the 3′ end of the nucleic acid molecule, for example. Theadditional sequence is not particularly limited. The length of theadditional sequence is not particularly limited, and is, for example, 1-to 200-mer, 1- to 50-mer, 1- to 25-mer, or 18-to 24-mer. The buildingblocks of the additional sequence are, for example, nucleotide residues,examples of which include deoxyribonucleotide residues andribonucleotide residues. The additional sequence is not particularlylimited, and examples thereof include polynucleotides such as a DNAconsisting of deoxyribonucleotide residues and a DNA including aribonucleotide residue(s). Specific examples of the additional sequenceinclude poly(dT) and poly(dA).

The α-amylase nucleic acid molecule of the present invention can be usedin the state where it is immobilized on a carrier, for example. It ispreferable to immobilize either the 5′ end or the 3′ end, morepreferably the 3′ end of the α-amylase nucleic acid molecule of thepresent invention, for example. When the α-amylase nucleic acid moleculeof the present invention is immobilized, the α-amylase nucleic acidmolecule may be immobilized either directly or indirectly on thecarrier, for example. In the latter case, it is preferable to immobilizethe α-amylase nucleic acid molecule via the additional sequence, forexample.

The method for producing the α-amylase nucleic acid molecule of thepresent invention is not particularly limited. For example, theα-amylase nucleic acid molecule of the present invention can besynthesized by known methods such as: nucleic acid synthesis methodsutilizing chemical synthesis; and genetic engineering procedures.

The α-amylase nucleic acid molecule of the present invention exhibitsbinding properties to the α-amylase, as mentioned above. Thus, use ofthe α-amylase nucleic acid molecule of the present invention is notparticularly limited, as long as it is the use utilizing the bindingproperties of the α-amylase nucleic acid molecule to the α-amylase. Theα-amylase nucleic acid molecule of the present invention can be used invarious methods as an alternative to, e.g., an antibody against theα-amylase.

(α-amylase Analysis Sensor)

The α-amylase analysis sensor of the present invention is a sensor foranalyzing α-amylase and includes the α-amylase-binding nucleic acidmolecule of the present invention. It is only required that theα-amylase analysis sensor of the present invention includes theα-amylase-binding nucleic acid molecule of the present invention, andother configurations, conditions, etc. are not particularly limited. Byusing the α-amylase analysis sensor of the present invention, theα-amylase can be detected by, for example, causing the α-amylase nucleicacid molecule to bind to the α-amylase. The description of theα-amylase-binding nucleic acid molecule of the present invention can beincorporated in the description of the α-amylase analysis sensor of thepresent invention by reference, for example.

The α-amylase analysis sensor of the present invention may be configuredso that, for example, it further includes a carrier, and theα-amylase-binding nucleic acid molecule is disposed on the carrier.Preferably, the α-amylase-binding nucleic acid molecule is immobilizedon the carrier. The immobilization of the α-amylase-binding nucleic acidmolecule on the carrier is as described above, for example. The methodfor using the α-amylase analysis sensor of the present invention is notparticularly limited, and the description of the α-amylase nucleic acidmolecule of the present invention and the following description of themethod for analyzing α-amylase of the present invention can beincorporated in the description of the α-amylase analysis sensor of thepresent invention by reference.

(Method for Analyzing α-amylase)

The method for analyzing α-amylase of the present invention includes thestep of causing a specimen and a nucleic acid molecule to come intocontact with each other to detect α-amylase in the specimen, the nucleicacid molecule is the α-amylase-binding nucleic acid molecule of thepresent invention, and in the detection step, the nucleic acid moleculeis caused to bind to the α-amylase in the specimen, and the α-amylase inthe specimen is detected by detecting the binding. The method foranalyzing α-amylase of the present invention is characterized in that ituses the α-amylase nucleic acid molecule of the present invention, andother steps, conditions, etc. are not particularly limited. In themethod for analyzing α-amylase of the present invention, the α-amylaseanalysis sensor of the present invention may be used as the α-amylasenucleic acid molecule of the present invention. The descriptions of theα-amylase-binding nucleic acid molecule and the α-amylase analysissensor of the present invention can be incorporated in the descriptionof the method for analyzing α-amylase of the present invention byreference, for example.

The nucleic acid molecule of the present invention specifically binds toα-amylase. Thus, according to the present invention, it is possible tospecifically detect α-amylase in a specimen by detecting the bindingbetween the α-amylase and the nucleic acid molecule, for example.Specifically, since the present invention can analyze the presence orabsence or the amount of α-amylase in a specimen, for example, it can besaid that the present invention can also perform qualitative orquantitative analysis of the α-amylase.

In the present invention, the specimen is not particularly limited.Examples of the specimen include saliva, urine, plasma, and serum.

The specimen may be a liquid specimen or a solid specimen, for example.The specimen is preferably a liquid specimen from the viewpoint of easeof handling because the liquid specimen can be caused to come intocontact with the nucleic acid molecule more easily, for example. In thecase of the solid specimen, a liquid mixture, a liquid extract, asolution, or the like of the solid specimen prepared using a solvent maybe used, for example. The solvent is not particularly limited, and maybe water, physiological saline, or a buffer solution, for example.

The above-described detection step includes, for example: a contact stepof causing the specimen and the nucleic acid molecule to come intocontact with each other to cause the nucleic acid molecule to bind tothe α-amylase in the specimen; and a binding detection step of detectingthe binding between the α-amylase and the nucleic acid molecule. Thedetection step may further include, for example, the step of analyzingthe presence or absence or the amount of the α-amylase in the specimenon the basis of the result obtained in the binding detection step.

In the contact step, the method for causing the specimen and the nucleicacid molecule to come into contact with each other is not particularlylimited. The contact between the specimen and the nucleic acid moleculepreferably is achieved in a liquid, for example. The liquid is notparticularly limited, and may be, for example, water, physiologicalsaline, or a buffer solution.

In the contact step, the conditions under which the contact between thespecimen and the nucleic acid molecule is caused are not particularlylimited. The contact temperature is, for example, 4° C. to 37° C., or18° C. to 25° C., and the contact time is, for example, 10 to 120minutes or 30 to 60 minutes.

In the contact step, the nucleic acid molecule may be an immobilizednucleic acid molecule immobilized on a carrier or an unimmobilizednucleic acid molecule in a free state, for example. In the latter case,the nucleic acid molecule is caused to come into contact with thespecimen in a container, for example. The nucleic acid molecule ispreferably the immobilized nucleic acid molecule from the viewpoint offavorable handleability, for example. The carrier is not particularlylimited, and may be, for example, a substrate, beads, or a container.The container may be a microplate or a tube, for example. Theimmobilization of the nucleic acid molecule is as described above, forexample.

The binding detection step is the step of detecting the binding betweenthe α-amylase in the specimen and the nucleic acid molecule, asmentioned above. By detecting the presence or absence of the bindingbetween the α-amylase and the nucleic acid molecule, it is possible toanalyze the presence or absence of the α-amylase in the specimen(qualitative analysis), for example. Also, by detecting the degree ofthe binding (the binding amount) of the α-amylase to the nucleic acidmolecule, it is possible to analyze the amount of the α-amylase in thespecimen (quantitative analysis), for example.

In the case where the binding between the α-amylase and the nucleic acidmolecule cannot be detected, it can be determined that no α-amylase ispresent in the specimen. In the case where the binding is detected, itcan be determined that the α-amylase is present in the specimen.

The method for analyzing the binding between the α-amylase and thenucleic acid molecule is not particularly limited. A conventionallyknown method for detecting the binding between substances may beemployed as the method, for example, and specific examples of the methodinclude the above-described SPR. Detection of the binding may bedetection of a complex of the α-amylase and the nucleic acid molecule,for example.

(α-amylase Detection Kit)

A α-amylase detection kit of the present invention includes theα-amylase-binding nucleic acid molecule of the present invention. It isonly required that the α-amylase detection kit of the present inventionincludes the α-amylase-binding nucleic acid molecule of the presentinvention, and other configurations, conditions, etc. are notparticularly limited. By using the α-amylase detection kit of thepresent invention, it is possible to perform the detection and the likeof the α-amylase as mentioned above, for example. The descriptions ofthe α-amylase-binding nucleic acid molecule, the α-amylase analysissensor, and the method for analyzing α-amylase of the present inventioncan be incorporated in the description of the α-amylase detection kit byreference, for example.

The α-amylase detection kit of the present invention may include theα-amylase analysis sensor of the present invention as the α-amylasenucleic acid molecule of the present invention, for example. Theα-amylase detection kit of the present invention may further include anycomponent(s) in addition to the α-amylase nucleic acid molecule of thepresent invention, for example. Examples of the component include theabove-described carrier, a buffer solution, and instructions for use.

(BDN4A-binding Nucleic Acid Molecule)

The β-defensin (BDN)4A-binding nucleic acid molecule (hereinafter alsoreferred to as “BDN4A nucleic acid molecule”) of the present inventionincludes the following polynucleotide (b):

(b) a polynucleotide (b1):(b1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:4 to 6.

The descriptions of the α-amylase-binding nucleic acid molecule, theα-amylase analysis sensor, the method for analyzing α-amylase, and theα-amylase detection kit can be incorporated in the description of theBDN4A-binding nucleic acid molecule of the present invention byreference, by, for example, reading “α-amylase” as “BDN4A”, reading“(a)” as “(b)”, reading “(a1)” as “(b1)”, reading “(a2)” as “(b2)”,reading “(a3)” as “(b3)”, reading “(a4)” as “(b4)”, and reading “SEQ IDNOs: 1 and 11 to 16” as “SEQ ID NOs: 4 to 6”, unless otherwisespecifically stated. The same applies to the descriptions of the BDN4Aanalysis sensor, the method for analyzing BDN4A, and the BDN4A detectionkit, to be described below.

The BDN4A nucleic acid molecule of the present invention can bind toBDN4A, as mentioned above. The BDN4A is not particularly limited, andthe BDN4A may be derived from a human or a non-human animal, forexample. Examples of the non-human animal include mice, rats, monkeys,rabbits, dogs, cats, horses, cows, and pigs. Amino acid sequenceinformation on human BDN4A is registered under Accession No. 015263 inUniProt (http://www.uniprot.org/), for example.

In the present invention, the expression “binds to BDN4A” (andgrammatical variations thereof) is also referred to as “has bindingability to BDN4A” or “has binding activity to BDN4A”, for example. Thebinding between the BDN4A-binding nucleic acid molecule of the presentinvention and the BDN4A can be determined by surface plasmon resonance(SPR) analysis or the like, for example. The analysis can be performedusing ProteON (trade name, BioRad), for example. Since the BDN4A nucleicacid molecule of the present invention binds to BDN4A, it can be usedfor detection of the BDN4A, for example.

As mentioned above, the BDN4A nucleic acid molecule of the presentinvention includes the following polynucleotide (b):

(b) a polynucleotide (1):(b1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:4 to 6.

BDN4A-binding nucleic acid molecule 1 (SEQ ID NO: 4)5′-GGTTACACGAGCCGCACATTTCTATTTTTACGGGGTATAGTTCTCTGAGGAGGAGTTCCCAGGCGAAGTTGTTATC-3′BDN4A-binding nucleic acid molecule 2 (SEQ ID NO: 5)5′-CGAGCCGCACATTTCTATTTTTACGGGGTATAGTTCTCTGAGGAGGAGTTCCCAGGCGAAGTTGTTATC-3′ BDN4A-binding nucleic acid molecule 3(SEQ ID NO: 6) 5′-GGTTACACGAGCCGCACATTTCACCGTGATAGTTCTCTGAGGAGGACTTCTAGAGTTCCCAGGCGAAGTTGTTATC-3′

The polynucleotide (b) above also includes, for example, the meaning ofthe polynucleotide of (b2), (b3), or (b4) below:

(b2) a polynucleotide consisting of a base sequence obtained bydeletion, substitution, insertion, and/or addition of one or more basesin any of the base sequences of the polynucleotide (b1) and binds to theBDN4A.(b3) a polynucleotide consisting of a base sequence having at least 80%sequence identity to any of the base sequences of the polynucleotide(b1) and binds to the BDN4A.(b4) a polynucleotide consisting of a base sequence complementary to apolynucleotide hybridizing to any of the base sequences of thepolynucleotide (b1) under stringent conditions and binds to the BDN4A.

(BDN4A Analysis Sensor)

The β-defensin (BDH)4A analysis sensor of the present invention is asensor for analyzing β-defensin (BDN)4A and includes the BDN4A-bindingnucleic acid molecule of the present invention. It is only required thatthe BDN4A analysis sensor of the present invention includes theBDN4A-binding nucleic acid molecule of the present invention, and otherconfigurations, conditions, etc. are not particularly limited. By usingthe BDN4A analysis sensor of the present invention, the BDN4A can bedetected by, for example, causing the BDN4A nucleic acid molecule tobind to the BDN4A. The description of the BDN4A-binding nucleic acidmolecule of the present invention can be incorporated in the descriptionof the BDN4A analysis sensor of the present invention by reference, forexample. The method for using the BDN4A analysis sensor of the presentinvention is not particularly limited, and the description of theBDN4A-binding nucleic acid molecule of the present invention and thefollowing description of the method for analyzing BDN4A of the presentinvention can be incorporated in the description of the BDN4A analysissensor of the present invention by reference.

(Method for Analyzing BDN4A)

The method for analyzing β-defensin (BDH)4A of the present inventionincludes the step of causing a specimen and a nucleic acid molecule tocome into contact with each other to detect β-defensin (BDN)4A in thespecimen, the nucleic acid molecule is the BDN4A-binding nucleic acidmolecule of the present invention, and in the detection step, thenucleic acid molecule is caused to bind to the BDN4A in the specimen,and the BDN4A in the specimen is detected by detecting the binding. Themethod for analyzing BDN4A of the present invention is characterized inthat it uses the BDN4A-binding nucleic acid molecule of the presentinvention, and other steps, conditions, etc. are not particularlylimited. In the method for analyzing BDN4A of the present invention, theBDN4A analysis sensor of the present invention may be used as the BDN4Anucleic acid molecule of the present invention. The descriptions of theBDN4A-binding nucleic acid molecule and the BDN4A analysis sensor of thepresent invention can be incorporated in the description of the methodfor analyzing BDN4A of the present invention by reference, for example.

(BDN4A Detection Kit)

The β-defensin (BDN)4A detection kit of the present invention includesthe BDN4A-binding nucleic acid molecule of the present invention. It isonly required that the BDN4A detection kit of the present inventionincludes the BDN4A-binding nucleic acid molecule of the presentinvention, and other configurations, conditions, etc. are notparticularly limited. By using the BDN4A detection kit of the presentinvention, it is possible to perform the detection and the like of theBDN4A as mentioned above, for example. The descriptions of theBDN4A-binding nucleic acid molecule, the BDN4A analysis sensor, and themethod for analyzing BDN4 of the present invention can be incorporatedin the description of the BDN4A detection kit of the present inventionby reference.

(Lysozyme-binding Nucleic Acid Molecule)

The lysozyme binding nucleic acid molecule (hereinafter also referred toas a “lysozyme nucleic acid molecule”) of the present invention includesthe following polynucleotide (1):

(1) a polynucleotide (11):(11) a polynucleotide consisting of any of base sequences of SEQ ID NOs:7 to 9.

The descriptions of the α-amylase-binding nucleic acid molecule, theα-amylase analysis sensor, the method for analyzing α-amylase, and theα-amylase detection kit can be incorporated in the description of thelysozyme-binding nucleic acid molecule of the present invention byreference, by, for example, reading “α-amylase” as “lysozyme”, reading“(a)” as “(1)”, reading “(a1)” as “(11)”, reading “(a2)” as “(12)”,reading “(a3)” as “(13)”, reading “(a4)” as “(14)”, and reading “SEQ IDNOs: 1 and 11 to 16” as “SEQ ID NOs: 7 to 9”, unless otherwisespecifically stated. The same applies to the descriptions of thelysozyme analysis sensor, the method for analyzing lysozyme, and thelysozyme detection kit, to be described below.

The lysozyme nucleic acid molecule of the present invention can bind tolysozyme, as mentioned above. The lysozyme is not particularly limited,and the lysozyme may be derived from a human or a non-human animal, forexample. Examples of the non-human animal include mice, rats, monkeys,rabbits, dogs, cats, horses, cows, and pigs. Amino acid sequenceinformation on human lysozyme is registered under Accession No. P61626in UniProt (http://www.uniprot.org/), for example.

In the present invention, the expression “binds to lysozyme” (andgrammatical variations thereof) is also referred to as “has bindingability to lysozyme” or “has binding activity to lysozyme”, for example.The binding between the lysozyme nucleic acid molecule of the presentinvention and the lysozyme can be determined by surface plasmonresonance (SPR) analysis or the like, for example. The analysis can beperformed using ProteON (trade name, BioRad), for example. Since thelysozyme nucleic acid molecule of the present invention binds tolysozyme, it can be used for detection of the lysozyme, for example.

As mentioned above, the lysozyme nucleic acid molecule of the presentinvention includes the following polynucleotide (l):

(l) a polynucleotide (l1):(l1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:7 to 9.

Lysozyme-binding nucleic acid molecule 1 (SEQ ID NO: 7)5′-GGTTACACGAGCCGCACATTTCTAACGGGAACTTCAACCCATACAGTCTTTTGAGTTCCCAGGCGAAGTTGTTATC-3′Lysozyme-binding nucleic acid molecule 2 (SEQ ID NO: 8)5′-CGAGCCGCACATTTCTAACGGGAACTTCAACCCATACAGTCTTT TGAGTTCCC-3′Lysozyme-binding nucleic acid molecule 3 (SEQ ID NO: 9)5′-GGTTACACGAGCCGCACATTTCTTTACTCCGGAACCCATACAGTCTTTTCCGGAGTTCCCAGGCGAAGTTGTTATC-3′

The polynucleotide (l) above also includes, for example, the meaning ofthe polynucleotide of (l2), (l3), or (l4) below:

(l2) a polynucleotide consisting of a base sequence obtained bydeletion, substitution, insertion, and/or addition of one or more basesin any of the base sequences of the polynucleotide (l1) and binds to thelysozyme.(l3) a polynucleotide consisting of a base sequence having at least 80%sequence identity to any of the base sequences of the polynucleotide(l1) and binds to the lysozyme.(l4) a polynucleotide consisting of a base sequence complementary to apolynucleotide hybridizing to any of the base sequences of thepolynucleotide (l1) under stringent conditions and binds to thelysozyme.

(Lysozyme Analysis Sensor)

The lysozyme analysis sensor of the present invention is a sensor foranalyzing lysozyme and includes the lysozyme-binding nucleic acidmolecule of the present invention. It is only required that the lysozymeanalysis sensor of the present invention includes the lysozyme-bindingnucleic acid molecule of the present invention, and otherconfigurations, conditions, etc. are not particularly limited. By usingthe lysozyme analysis sensor of the present invention, the lysozyme canbe detected by, for example, causing the lysozyme nucleic acid moleculeto bind to the lysozyme. The description of the lysozyme-binding nucleicacid molecule of the present invention can be incorporated in thedescription of the lysozyme analysis sensor of the present invention byreference, for example. The method for using the lysozyme analysissensor of the present invention is not particularly limited, and thedescription of the lysozyme-binding nucleic acid molecule of the presentinvention and the following description of the method for analyzinglysozyme of the present invention can be incorporated in the descriptionof the lysozyme analysis sensor of the present invention by reference.

(Method for Analyzing Lysozyme)

The method for analyzing lysozyme of the present invention includes thestep of causing a specimen and a nucleic acid molecule to come intocontact with each other to detect lysozyme in the specimen, the nucleicacid molecule is the lysozyme-binding nucleic acid molecule of thepresent invention, and in the detection step, the nucleic acid moleculeis caused to bind to the lysozyme in the specimen, and the lysozyme inthe specimen is detected by detecting the binding.

The method for analyzing lysozyme of the present invention ischaracterized in that it uses the lysozyme-binding nucleic acid moleculeof the present invention, and other steps, conditions, etc. are notparticularly limited. In the method for analyzing lysozyme of thepresent invention, the lysozyme analysis sensor of the present inventionmay be used as the lysozyme nucleic acid molecule of the presentinvention. The descriptions of the lysozyme-binding nucleic acidmolecule and the lysozyme analysis sensor of the present invention canbe incorporated in the description of the method for analyzing lysozymeof the present invention by reference, for example.

(Lysozyme Detection Kit)

The lysozyme detection kit of the present invention includes thelysozyme-binding nucleic acid molecule of the present invention. It isonly required that the lysozyme detection kit of the present inventionincludes the lysozyme-binding nucleic acid molecule of the presentinvention, and other configurations, conditions, etc. are notparticularly limited. By using the lysozyme detection kit of the presentinvention, it is possible to perform the detection and the like of thelysozyme as mentioned above, for example. The descriptions of thelysozyme-binding nucleic acid molecule, the lysozyme analysis sensor,and the method for analyzing lysozyme of the present invention can beincorporated in the description of the lysozyme detection kit byreference, for example.

(LDHS-binding Nucleic Acid Molecule)

The lactate dehydrogenase (LDH)5-binding nucleic acid molecule(hereinafter also referred to as a “LDH5 nucleic acid molecule”) of thepresent invention includes the following polynucleotide (d):

(d) a polynucleotide (d1):(d1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:17 to 20.

The descriptions of the α-amylase-binding nucleic acid molecule, theα-amylase analysis sensor, the method for analyzing α-amylase, and theα-amylase detection kit can be incorporated in the description of theLDH5-binding nucleic acid molecule of the present invention byreference, by, for example, reading “α-amylase” as “LDH5”, reading “(a)”as “(d)”, reading “(a1)” as “(d1)”, reading “(a2)” as “(d2)”, reading“(a3)” as “(d3)”, reading “(a4)” as “(d4)”, and reading “SEQ ID NOs: 1and 11 to 16” as “SEQ ID NOs: 17 to 20”, unless otherwise specificallystated. The same applies to the descriptions of the LDH5 analysissensor, the method for analyzing LDH5, and the LDH5 detection kit, to bedescribed below.

The LDH5 nucleic acid molecule of the present invention can bind to LDH5as mentioned above. The LDH5 is not particularly limited, and the LDH5may be derived from a human or a non-human animal, for example. Examplesof the non-human animal include mice, rats, monkeys, rabbits, dogs,cats, horses, cows, and pigs. Amino acid sequence information on humanLDH5 is registered under Accession No. P00338 in UniProt(http://www.uniprot.org/), for example.

In the present invention, the expression “binds to LDH5” (andgrammatical variations thereof) is also referred to as “has bindingability to LDH5” or “has binding activity to LDH5”, for example. Thebinding between the LDH5 nucleic acid molecule of the present inventionand the LDH5 can be determined by surface plasmon resonance (SPR)analysis or the like, for example. The analysis can be performed usingProteON (trade name, BioRad), for example. Since the LDH5 nucleic acidmolecule of the present invention binds to LDH5, it can be used fordetection of the LDH5, for example.

As mentioned above, the LDH5 nucleic acid molecule of the presentinvention includes the following polynucleotide (d):

(d) a polynucleotide (d1):(d1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:17 to 20.

LDH5-binding nucleic acid molecule 1 (SEQ ID NO: 17)5′-GGAATTGACACCTCGCCGTTTATGCTGCTGGCTCGTGAGACGGATATCAGGTCTCCTAAGGCTGGCTGGCTACTATAC-3′LDH5-binding nucleic acid molecule 2 (SEQ ID NO: 18)5′-GGAATTGACACCTCGCCGTTTATGAGAGGGAGATCATCTCTCTGGCGGACACAACCTAAGGCTGGCTGGCTACTATAC-3′LDH5-binding nucleic acid molecule 3 (SEQ ID NO: 19)5′-ACCTCGCCGTTTATGCTGCTGGCTCGTGAGACGGATATCAGGTC TCCTAAGGCTGGC-3′LDH5-binding nucleic acid molecule 4 (SEQ ID NO: 20)5′-TGCTGCTGGCTCGTGAGACGGATATCAGGTCTCCTAAGGCTGG C-3′

The polynucleotide (d) above also includes, for example, the meaning ofthe polynucleotide of (d2), (d3), or (d4) below:

(d2) a polynucleotide consisting of a base sequence obtained bydeletion, substitution, insertion, and/or addition of one or more basesin any of the base sequences of the polynucleotide (d1) and binds to theLDH5.(d3) a polynucleotide consisting of a base sequence having at least 80%sequence identity to any of the base sequences of the polynucleotide(d1) and binds to the LDH5.(d4) a polynucleotide consisting of a base sequence complementary to apolynucleotide hybridizing to any of the base sequences of thepolynucleotide (d1 ) under stringent conditions and binds to the LDH5.

(LDH5 Analysis Sensor)

The LDH5 analysis sensor of the present invention is a sensor foranalyzing LDH5 and includes the LDH5-binding nucleic acid molecule ofthe present invention. It is only required that the LDH5 analysis sensorof the present invention includes the LDH5-binding nucleic acid moleculeof the present invention, and other configurations, conditions, etc. arenot particularly limited. By using the LDH5 analysis sensor of thepresent invention, the LDH5 can be detected by, for example, causing theLDH5 nucleic acid molecule to bind to the LDH5. The description of theLDH5-binding nucleic acid molecule of the present invention can beincorporated in the description of the LDH5 analysis sensor of thepresent invention by reference, for example. The method for using theLDH5 analysis sensor of the present invention is not particularlylimited, and the description of the LDH5-binding nucleic acid moleculeof the present invention and the following description of the method foranalyzing LDH5 of the present invention can be incorporated in thedescription of the LDH5 analysis sensor of the present invention byreference.

(Method for Analyzing LDH5)

The method for analyzing LDH5 of the present invention includes the stepof causing a specimen and a nucleic acid molecule to come into contactwith each other to detect LDH5 in the specimen, the nucleic acidmolecule is the LDH5-binding nucleic acid molecule of the presentinvention, and in the detection step, the nucleic acid molecule iscaused to bind to the LDH5 in the specimen, and the LDH5 in the specimenis detected by detecting the binding. The method for analyzing LDH5 ofthe present invention is characterized in that it uses the LDH5-bindingnucleic acid molecule of the present invention, and other steps,conditions, etc. are not particularly limited. In the method foranalyzing LDH5 of the present invention, the LDH5 analysis sensor of thepresent invention may be used as the LDH5 nucleic acid molecule of thepresent invention. The descriptions of the LDH5-binding nucleic acidmolecule and the LDH5 analysis sensor of the present invention can beincorporated in the description of the method for analyzing LDH5 of thepresent invention by reference, for example.

(LDH5 Detection Kit)

The LDH5 detection kit of the present invention includes theLDH5-binding nucleic acid molecule of the present invention. It is onlyrequired that the LDH5 detection kit of the present invention includesthe LDH5-binding nucleic acid molecule of the present invention, andother configurations, conditions, etc. are not particularly limited. Byusing the LDH5 detection kit of the present invention, it is possible toperform the detection and the like of the LDH5 as mentioned above, forexample. The descriptions of the LDH5-binding nucleic acid molecule, theLDH5 analysis sensor, and the method for analyzing LDH5 of the presentinvention can be incorporated in the description of the LDH5 detectionkit of the present invention by reference.

(IL-6-binding nucleic acid molecule)

The interleukin (IL)-6-binding nucleic acid molecule (hereinafter alsoreferred to as “IL-6 nucleic acid molecule”) of the present inventionincludes the following polynucleotide (i):

(i) a polynucleotide (i1):(i1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:21 and 22.

The descriptions of the α-amylase-binding nucleic acid molecule, theα-amylase analysis sensor, the method for analyzing α-amylase, and theα-amylase detection kit can be incorporated in the description of theIL-6-binding nucleic acid molecule of the present invention byreference, by, for example, reading “α-amylase” as “IL-6”, reading “(a)”as “(i)”, reading “(a1)” as “(i1)”, reading “(a2)” as “(i2)”, reading“(a3)” as “(i3)”, reading “(a4)” as “(i4)”, and reading “SEQ ID NOs: 1and 11 to 16” as “SEQ ID NOs: 21 and 22”, unless otherwise specificallystated. The same applies to the descriptions of the IL-6 analysissensor, the method for analyzing IL-6, and the IL-6 detection kit, to bedescribed below.

The IL-6 nucleic acid molecule of the present invention can bind toIL-6, as mentioned above. The IL-6 is not particularly limited, and theIL-6 may be derived from a human or a non-human animal, for example.Examples of the non-human animal include mice, rats, monkeys, rabbits,dogs, cats, horses, cows, and pigs. Amino acid sequence information onhuman IL-6 is registered under Accession No. P05231 in UniProt(http://www.uniprot.org/), for example.

In the present invention, the expression “binds to IL-6” (andgrammatical variations thereof) is also referred to as “has bindingability to IL-6” or “has binding activity to IL-6”, for example. Thebinding between the IL-6 nucleic acid molecule of the present inventionand the IL-6 can be determined by surface plasmon resonance (SPR)analysis or the like, for example. The analysis can be performed usingProteON (trade name, BioRad), for example. Since the IL-6 nucleic acidmolecule of the present invention binds to IL-6, it can be used fordetection of the IL-6, for example.

As mentioned above, the IL-6 nucleic acid molecule of the presentinvention includes the following polynucleotide (i):

(i) a polynucleotide (i1):(i1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:21 and 22.

IL-6-binding nucleic acid molecule 1 (SEQ ID NO: 21)5′-GGAATTGACACCTCGCCGTTTATGAGTTCAATGGTATTGTATCGACTCTTCTCGCCTAAGGCTGGCTGGCTACTATAC-3′IL-6-binding nucleic acid molecule 2 (SEQ ID NO: 22)5′-ACCTCGCCGTTTATGAGTTCAATGGTATTGTATCGACTCTTCT C-3′

The polynucleotide (i) above also includes, for example, the meaning ofthe polynucleotide of (i2), (i3), or (i4) below.

(i2) a polynucleotide consisting of a base sequence obtained bydeletion, substitution, insertion, and/or addition of one or more basesin any of the base sequences of the polynucleotide (i1) and binds to theIL-6.(i3) a polynucleotide comprising a base sequence having 80% or moreidentity to the base sequence of any one of the above (i1) and binds toIL-6.(i4) a polynucleotide consisting of a base sequence complementary to apolynucleotide hybridizing to any of the base sequences of thepolynucleotide (i1) under stringent conditions and binds to the IL-6.

(IL-6 Analysis Sensor)

The IL-6 analysis sensor of the present invention is a sensor foranalyzing IL-6 and includes the IL-6-binding nucleic acid molecule ofthe present invention. It is only required that the IL-6 analysis sensorof the present invention includes the IL-6-binding nucleic acid moleculeof the present invention, and other configurations, conditions, etc. arenot particularly limited. By using the IL-6 analysis sensor of thepresent invention, the IL-6 can be detected by, for example, causing theIL-6 nucleic acid molecule to bind to the IL-6. The description of theIL-6-binding nucleic acid molecule of the present invention can beincorporated in the description of the IL-6 analysis sensor of thepresent invention by reference, for example. The method for using theIL-6 analysis sensor of the present invention is not particularlylimited, and the description of the IL-6-binding nucleic acid moleculeof the present invention and the following description of the method foranalyzing IL-6 of the present invention can be incorporated in thedescription of the IL-6 analysis sensor of the present invention byreference.

(Method for Analyzing IL-6)

The method for analyzing IL-6 of the present invention includes the stepof causing a specimen and a nucleic acid molecule to come into contactwith each other to detect IL-6 in the specimen, the nucleic acidmolecule is the IL-6-binding nucleic acid molecule of the presentinvention, and in the detection step, the nucleic acid molecule iscaused to bind to the IL-6 in the specimen, and the IL-6 in the specimenis detected by detecting the binding. The method for analyzing IL-6 ofthe present invention is characterized in that it uses the IL-6-bindingnucleic acid molecule of the present invention, and other steps,conditions, etc. are not particularly limited. In the method foranalyzing IL-6 of the present invention, the IL-6 analysis sensor of thepresent invention may be used as the IL-6 nucleic acid molecule of thepresent invention. The descriptions of the IL-6-binding nucleic acidmolecule and the IL-6 analysis sensor of the present invention can beincorporated in the description of the method for analyzing IL-6 of thepresent invention by reference, for example.

(IL-6 Detection Kit)

The IL-6 detection kit of the present invention includes theIL-6-binding nucleic acid molecule of the present invention. It is onlyrequired that the IL-6 detection kit of the present invention includesthe IL-6-binding nucleic acid molecule of the present invention, andother configurations, conditions, etc. are not particularly limited. Byusing the IL-6 detection kit of the present invention, it is possible toperform the detection and the like of the IL-6 as mentioned above, forexample. The descriptions of the IL-6-binding nucleic acid molecule, theIL-6 analysis sensor, and the method for analyzing IL-6 of the presentinvention can be incorporated in the description of the IL-6 detectionkit of the present invention by reference.

EXAMPLES

The present invention is described more specifically below withreference to examples. It is to be noted, however, that the scope of thepresent invention is not limited by these examples. Commerciallyavailable reagents in the examples were used in accordance with theirprotocols, unless otherwise stated.

Example 1

MK1 to MK4 were prepared by the following synthesis examples.

Electrospray ionization mass spectrometry (ESI-MS) was performed using amass spectrometer (API2000, vendor: Applied Biosystems) in positive ornegative ion mode. ¹H NMR spectra were obtained using a nuclear magneticresonance instrument (JNM-ECS400, manufactured by JEOL). Chemical shiftsare expressed as relative δ (ppm) to the internal standard,tetramethylsilane (Me₄Si). Ion-exchange chromatography was performedusing a chromatographic system (ECONO system, manufactured by Bio-Rad).In the ion-exchange chromatography, a glass column (φ25×500 mm) packedwith diethylaminoethyl (DEAE) A-25-Sephadex (manufactured byAmershambiosciences) was used.

Synthesis Example 1 Synthesis of MK1

AZ6 (290 mg, 9.06×10⁻⁴ mol) was dried in vacuo and dissolved in dry-DMF(N,N-dimethylformamide, 3 mL). To this solution, HOBt.H₂O(1-hydroxybenzotriazole monohydrate, 176 mg, 1.15×10⁻⁵ mol, 1.2 eq.),PyBOP® (hexafluorophosphoric acid(benzotriazole-1-yloxy)tripyrrolidinophosphonium, 579 mg, 1.15×10⁻⁵ mol,1.2 eq.), and DIPEA (N,N-diisopropylethylamine, 4.6 mL, 2.72×10⁻² mol,30 eq.) were added, and NK1 (493 mg, 9.48×10 mol, 1.1 eq.) dissolved indry-DMF (1 mL) was further added and stirred. After 40 minutes from theinitiation of the stirring, the solvent was distilled off under reducedpressure, and a residue was dissolved in water, and a precipitate wascollected by suction filtration. The filtrate was roughly purified byreversed-phase column chromatography, to give MK1.

Physical properties of MK1 are shown below.Yield amount: 261 mg, Yield: 60%ESI-MS (positive ion mode) m/z, found =481.2, calculated for [(M+H)+]=481.2 found =503.1, calculated for [(M+Na)+] =503.2

¹HNMR (400 MHz, DMSO-d6) δ8.22 (1H, m), 8.11 (1H, s), 8.10 (1H, s), 7.87(1H, s), 7.63 (1H, d), 6.52 (1H, q), 6.35 (1H, d), 5.27 (1H, s), 3.82(1H, m), 2.18 (1H, m) Synthesis Example 2 Synthesis of MK2

MK1 (108 mg, 2.25×10⁴ mol) was dried in vacuo, and the atmosphere wasreplaced with Ar (Argon). Subsequently, azeotropy between the MK1 anddry-DMF was caused twice (the first time: 40 mL, the second time: 4 mL),and azeotropy between the MK1 and dry-MeCN (acetonitrile) was causedthree times (the first time: 9 mL, the second time: 5 mL, the thirdtime: 5 mL). The resultant azeotrope was suspended in dry-Trimethylphosphate (6 mL), and thereafter, dry-Tributhyl amine (130 μL, 5.44×10⁴mol, 2.5 eq.) was added thereto. Then, phosphoryl chloride (42 μL,4.50×10⁴ mol, 2 eq.) was added and stirred under ice cooling.

After 40 minutes from the initiation of the stirring, dry-Tributhylamine (250 μL, 1.05 ×10⁻³ mol, 5 eq.) and Phosphoryl chloride (84 μL,4.50×10⁴ mol, 4 eq.) were again added and stirred under ice cooling for1 hour. After the stirring, a cooled 1 mol/L TEAB (Triethylammoniumbicarbonate) buffer (5 mL) was added, stirred for 5 minutes, andquenched. Then, the solvent was distilled off under reduced pressure,crystallization was performed in Ether, and suction filtration wasperformed to obtain a yellow solid. The yellow solid was dissolved inwater, purified by anion-exchange column chromatography, andfreeze-dried, to give MK2. Physical properties of MK2 are shown below.

Yield: 30.0 μmol yield: 13.4%ESI-MS (negative ion mode) m/z, found =559.1, calculated for[(M−H)−]=559.2

Synthesis Example 3 Synthesis of MK3

MK2 (30.03 _(I)lmol) was dried in vacuo, and azeotropy between the MK2and dry-Pyridine (10 mL) was performed three times, and the azeotropewas dried in vacuo overnight. After the drying, the atmosphere wasreplaced with Ar, and the MK2 was dissolved in dry-DMF (2 mL) anddry-TEA (triethylamine, 28 μL, 1.98×10⁴ mol, 6.6 eq.). Further,Imidazole (16 mg, 14.02×10⁴ mol, 4 eq.), 2,2′-Dithiodipyridine (17 mg,7.72×10 mol, 1.6 eq.), and Triphenylphosphine (20 mg, 7.63×10⁴ mol, 1.6eq.) were added and stirred at room temperature. After 6.5 hours fromthe initiation of the stirring, the resultant reaction solution wasadded to a solution of Sodium perchlorate (39 mg, 3.19×10⁴ mol, 10 eq.)in dry-Acetone (18 mL), dry Ether (27 mL), and dry-TEA (2 mL), andallowed to stand at 4° C. for 30 minutes. The precipitate was decanted 5times with dry-Ether (12 mL) and was thereafter dried in vacuo, to giveMK3 as a crude.

Theoretical yield amount: 30.03 μmol

Synthesis Example 4 Synthesis of MK4

MK3 (30.03 μmol) was dried in vacuo, the atmosphere was replaced withAr, and then, azeotropy between the MK3 and dry-Pyridine (5 mL) wascaused twice, and the azeotrope was then suspended in dry-DMF (1 mL).Further, dry-n-Tributylamine (30 μL, 1.25×10⁴ mol, 4 eq.) and 0.5 mol/Ln-Tributylamine pyrophosphate in DMF (310 μL, 1.53×10⁴ mol, 5 eq.) wereadded to the suspension and then stirred at room temperature. After 6.5hours from the initiation of the stirring, a 1 mol/L TEAB buffer (5 mL)was added and stirred for 30 minutes, and then the solvent was distilledoff under reduced pressure. Water was added, an aqueous layer wasseparated with Ether twice, purified by anion-exchange columnchromatography, and freeze-dried, to give MK4. Physical properties ofMK4 are shown below.

Yield: 3.33 μmol, Yield: 11.1%ESI-MS (negative ion mode) m/z, found =719.0, calculated for[(M−H)−]=719.1

Example 2

The present example examined whether binding nucleic acid molecules thatbind to sIgA and binding nucleic acid molecules that bind to α-amylasecan be obtained using MK4.

(1) Binding Nucleic Acid Molecule

Binding nucleic acid molecules that bind to a target were obtained bythe SELEX method, except that candidate polynucleotides prepared byusing, in addition to deoxyribonucleotides containing thymine, guanine,and cytosine, respectively (dTTP, and dGTP, and dCTP, respectively), MK4as deoxyribonucleotide were used. Specifically, the binding nucleic acidmolecules were obtained in the following manner. sIgA (manufactured byMP Biomedicals, LLC-Cappel Products) or human salivary amylase(manufactured by Lee BioSolutions, Inc.) as the target was bound tobeads (Dynabeads MyOne Carboxylic Acid, manufactured by Invitrogen)according to the protocols attached to the products. After binding thetarget, the beads were washed with a selection buffer (SB Buffer: 40mmol/L HEPES, 125 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl₂, 0.01%Tween® 20, pH 7.5), whereby target beads were prepared. dsDNAs with MK4inserted therein were prepared using complementary strands with their 5′ends modified with biotin (forward (Fw) primer region-N30 (30bases)-reverse (Rv) primer region), forward primers and DNA polymerase(KOD Dash, manufactured by Toyobo Co., Ltd.), and dTTP, dGTP, dCTP andMK4. Subsequently, the dsDNAs were bound to the beads (Dynabeads MyOneCarboxylic Acid), and then, ss (single strand) DNAs were eluted with a0.02 mol/L NaOH aqueous solution. Furthermore, the NaOH aqueous solutionwas neutralized with a 0.08 mol/L hydrochloric acid aqueous solution.Thus, an ssDNA library was prepared. 20 pmol of the library was mixedwith 250 ₁.tg of the target beads at 25° C. for 15 minutes. Thereafter,the beads were washed with the SB buffer. Then, the ssDNAs bound to thebeads were eluted with a 7 mol/L urea aqueous solution. The elutedssDNAs were amplified by PCR using the forward primers and thebiotin-modified reverse primers. In the PCR, dTTP, adenine-containingdeoxyribonucleotide (dATP), dGTP, and dCTP were used asdeoxyribonucleotides. The obtained dsDNAs were bound to magnetic beads(Dynabeads MyOne SA Cl magnetic beads, manufactured by Invitrogen).Thereafter, forward strands were eluted with a 0.02 mol/L NaOH aqueoussolution and removed. After removing the forward strands, the magneticbeads were washed with the SB buffer. Using the magnetic beads with thecomplementary strands immobilized thereon, forward primers and DNApolymerase (KOD Dash, manufactured by Toyobo Co., Ltd.), dTTP, dGTP,dCTP, and MK4, dsDNAs with MK4 inserted therein were prepared in theabove-described manner. Next, an ssDNA library was prepared by elutingforward strands with a 0.02 mol/L NaOH aqueous solution, and thislibrary was used in a subsequent round. Nucleic acid molecules that bindto sIgA or α-amylase were selected by performing eight rounds of thesame process. Thereafter, PCR was performed using forward primers andreverse primers without biotin modification. The obtained nucleic acidmolecules were subjected to sequencing using a sequencer (GS juniorsequencer, manufactured by Roche).

As a result, a binding nucleic acid molecule consisting of the basesequence of SEQ ID NO: 1 below was obtained as the binding nucleic acidmolecule that binds to α-amylase, and a binding nucleic acid moleculeconsisting of the base sequence of SEQ ID NO: 2 below was obtained asthe binding nucleic acid molecule that binds to sIgA. In the basesequences of SEQ ID NOs: 1 and 2, the underlined bases A are MK4.

α-amylase-binding nucleic acid molecule 1 (SEQ ID NO: 1)5′-GGTTTGGACGCAATCTCCCTAATCTAGTGACGAAAATGTACGAGGGGGTCATTTGAAACTACAATGGGCGGGCTTATC-3′ sIgA-binding nucleic acid molecule(SEQ ID NO: 2) 5′-GGTTTGGACGCAATCTCCCTAATCAAGCCACGGAGAGTCCGAGGTGACCATTAAGCAGGAAACTACAATGGGCGGGCTTA-3′

(2) Examination of Binding by Surface Plasmon Resonance (SPR)

The binding between the α-amylase-binding nucleic acid molecule 1 andα-amylase and the binding between the sIgA-binding nucleic acid moleculeand sIgA were measured under the following SPR conditions. Theα-amylase-binding nucleic acid molecule 1 and sIgA-binding nucleic acidmolecule adapted so that a 20-mer poly(dT) was added to the 3′ ends wereeach used as the following ligand 2. Further, as control 1 and control2, examination of the binding was performed in the same manner exceptthat, in control 1, bovine serum albumin (BSA) was used as the followinganalyte, and in control 2, chromogranin A (CgA, manufactured by CreativeBioMart) was used as the following analyte in a system for examining thebinding of the α-amylase-binding nucleic acid molecules 1 and theα-amylase was used as the following analyte in a system for examiningthe binding of the sIgA-binding nucleic acid molecules.

(SPR Measurement Conditions)

Measurement device: ProteOn™ XPR36 (manufactured by BioRad)Measurement chip: ProteOn™ NLC Sensor Chip (manufactured by BioRad)Ligand 1: poly(dA) (20-mer) with the 5′ end thereof being modified withbiotin: 5 μmol/LBuffer: 40 mmol/L HEPES, 125 mmol/L NaCl, 1 mmol/L MgCl₂, 5 mmol/L KCl,0.01%

Tween® 20, pH 7.4

Ligand 2: buffer containing a binding nucleic acid molecule with poly(T)(20-mer) added to the 3′ end at 200 nmol/LLigand flow rate: 25 μL/min, 80 secAnalyte: buffer containing a target at 400 nmol/LAnalyte flow rate: 50 μL/min

Contact Time: 120 sec Dissociation: 300 sec

-   -   sIgA: IgA (Secretory), Human (manufactured by MP Biomedicals,        LLC-Cappel Products, Catalogue number: #55905)    -   Amylase: α-amylase (manufactured by Lee Biosolutions, Catalogue        number: #120-10)    -   CgA: recombinant full length Human Chromogranin A (manufactured        by Creative BioMart, Catalog number: #CHGA 26904TH)    -   BSA: Bovine Serum Albumin (manufactured by SIGMA, Catalogue        number: #A7906)

The results of measuring the binding between the α-amylase-bindingnucleic acid molecule 1 and the α-amylase are shown in FIG. 1, and theresults of measuring the binding between the sIgA-binding nucleic acidmolecule and sIgA are shown in FIG. 2. FIG. 1 is a graph showing thebinding ability of the α-amylase-binding nucleic acid molecule 1 to theα-amylase. In FIG. 1, the horizontal axis indicates the time elapsedafter the injection of the ligand, and the vertical axis indicates therelative value (RU) of the binding force. As can be seen in FIG. 1, theα-amylase-binding nucleic acid molecule 1 bound to the α-amylase,whereas they did not bind to CgA or BSA.

Next, FIG. 2 is a graph showing the binding ability of the sIgA-bindingnucleic acid molecule to sIgA. In FIG. 2, the horizontal axis indicatesthe time elapsed after the injection of the ligand, and the verticalaxis indicates the relative value (RU) of the binding force. As can beseen in FIG. 2, the sIgA-binding nucleic acid molecules bind sIgA,whereas they do not bind to the α-amylase or BSA.

From these results, it was found that a binding nucleic acid moleculethat binds to α-amylase and a binding nucleic acid molecule that bindsto sIgA can be obtained using MK4, which is the nucleoside derivative ofthe present invention.

(3) Examination of Binding Force

The relative value (RU) of the binding force was measured in the samemanner as in the above item (2), except that the α-amylase-bindingnucleic acid molecule 1 having a 20-mer poly(T) added to its 3′ end wasused as the ligand 2 and that the concentration of the α-amylase as theanalyte was set to 5, 10, 20, 40, or 80 nmol/L. Also, the relative value(RU) of the binding force was measured in the same manner as in theabove item (2), except that the sIgA-binding nucleic acid moleculehaving a 20-mer poly(T) added to its 3′ end was used as the ligand 2 andthat the concentration of sIgA as the analyte was 12.5, 25, 50, 100, or200 nmol/L. Then, based on the relative values (RU) of the binding forcemeasured in the above, the dissociation constant between theα-amylase-binding nucleic acid molecule 1 and the α-amylase and thedissociation constant between the sIgA-binding nucleic acid molecule andsIgA were calculated. As a result, the dissociation constant between theα-amylase-binding nucleic acid molecules 1 and the α-amylase was 8.14nM, and the dissociation constant between the sIgA-binding nucleic acidmolecules and sIgA was 7.63 nM. These results demonstrate that both thebinding nucleic acid molecules have excellent binding ability to thetargets.

(4) Examination of Binding by Capillary Electrophoresis

Binding between the α-amylase-binding nucleic acid molecule 1 andα-amylase was measured by capillary electrophoresis performed under thefollowing conditions. The α-amylase-binding nucleic acid moleculeadapted so that the 5′ end thereof was labeled with a 20-mer TYE™ 665was used as the following clone. As a control, the measurement wasperformed in the same manner except that α-amylase was not added as thetarget.

(Conditions of Capillary Electrophoresis)

Measurement device: Cosmo-i SV1210 (Hitachi High-TechnologiesCorporation)Measurement chip: i-chip 12 (Hitachi Chemical Company, Ltd.)Electrophoresis gel: 0.6% (Hydroxypropyl)methyl cellulose, viscosity 2.600-5, 600 (manufactured by SIGMA, Catalogue number: #H7509)Gel dissolving buffer: 40 mmol/L HEPES (pH 7.5), 5 mmol/L KCl, 1 mmol/LMgCl₂Clone: solution containing 200 nmol/L amylase-binding nucleic acidmolecule with its 5′ end labeled with TYETM 665, 40 mmol/L HEPES (pH7.5), 125 mmol/L NaCl, 5 mmol/L KCl, and 1 mmol/L MgCl₂Target: solution containing 4 μmol/L amylase (a-Amylase-High Purity,Human, manufactured by Lee BioSolutions, Inc., Catalogue number:#120-10), 40 mmol/L HEPES (pH 7.5), 125 mmol/LNaCl, 5 mmol/L KCl, and 1 mmol/L MgCl₂Folding: 95° C., after 5 min, on ice 5 minMixing: after addition of target, room temperature (around 25° C.), 30min, 1000 rpmInjection voltage: 600 VInjection time: 120 secSeparation voltage: 350 V

Separation Time: 260 sec

The results obtained are shown in FIG. 3. FIG. 3 is a photograph showingthe results of capillary electrophoresis. In FIG. 3, the electrophoresistime is shown on the left side of the photograph, and the respectivelanes show, from the left, the result obtained regarding the control(without α-amylase) and the result obtained when the α-amylase was used.As can be seen in FIG. 3, the electrophoresis time in the presence ofthe α-amylase was longer than that in the control without α-amylase.From these results, it was found that the α-amylase-binding nucleic acidmolecule 1 binds to α-amylase.

(5) Examination of Binding by Pull-Down Assay

Beads 1 carrying α-amylase-binding nucleic acid molecule bound thereto(hereinafter, also referred to as “bound beads Al”) were prepared bybringing an α-amylase-binding nucleic acid molecule 1 with its 5′ endmodified with biotin into contact with streptavidin-modified beads(Dynabeads MyOne SA C1 magnetic beads, manufactured by Invitrogen).Next, the bound beads A1 were mixed with a saliva-containing SB buffer(saliva sample), and the resultant mixture was shaken at 1000 rpm for 60minutes at room temperature (around 25° C.).

After the shaking, the bound beads A1 were washed three times with theSB buffer. Then, the bound beads A1 were treated at 95° C. for 10minutes in the presence of the SDS buffer, whereby the α-amylase boundto the bound beads A1 was eluted. The composition of the SDS buffer wasas follows: 62.5 mmol/L Tris, 2% SDS, 5% sucrose, 0.002% Bromophenolblue, and 1% 2-mercaptoethanol.

The thus-obtained eluate was loaded onto a gel (PAGEL C5OL, manufacturedby ATTO), and electrophoresis was performed in the presence of anelectrophoresis buffer. The composition of the electrophoresis bufferwas as follows: 25 mmol/L Tris, 192 mmol/L glycine, and 0.1% SDS. Next,the gel after the electrophoresis was stained with a staining agent (GelCode, manufactured by Thermo SCIENTIFIC, Catalogue number: #24594), andimaged with ChemiDoc (manufactured by BioRad). Further, as control 1 andcontrol 2, imaging was performed in the same manner except that, incontrol 1, nucleic acid molecules that do not bind to α-amylase withtheir 5′ ends modified with biotin were used (control nucleic acidmolecules 1) and, in control 2, only α-amylase was used.

Control nucleic acid molecule 1 (SEQ ID NO: 3)5′-GGATACCTTAACGCCGCCTATTG-3′

The results obtained are shown in FIG. 4. FIG. 4 is a photograph showingthe results of the pull-down assay. In FIG. 4, the numerical values onthe left side of the photograph indicate molecular weights, and therespective lanes show, from the left, the results obtained regarding themolecular weight marker (M), the saliva sample (1), control 1 (C1), andcontrol 2 (AMY). As can be seen in FIG. 4, in the lane showing theresult of control 1, no band was observed at the same position (about 50kDa) as in the lane showing the result of control 2, whereas, in thelane showing the result regarding the saliva sample, a band was observedat the same electrophoretic mobility position as in the lane showing theresult of control 2. In other words, the binding of theα-amylase-binding nucleic acid molecules 1 to α-amylase was observed.From these results, it was found that the α-amylase-binding nucleic acidmolecule 1 binds to α-amylase.

Example 3

The present example examined whether a binding nucleic acid moleculethat binds to human β-defensin 4A and a binding nucleic acid moleculethat binds to human lysozyme can be obtained using MK4.

(1) Binding Nucleic Acid Molecule

The binding nucleic acid molecule that binds to human β-defensin 4A andthe binding nucleic acid molecule that binds to human lysozyme wereobtained in the same manner as in the above item (1) in Example 2,except that, instead of sIgA as the target, human β-defensin 4A(manufactured by Novoprotein Scientific Inc., Catalog number: #C127) orhuman lysozyme (manufactured by Novoprotein Scientific Inc., Catalognumber: #P61626) was used.

As a result, as the binding nucleic acid molecule that binds toβ-defensin (BDN)4A, the binding nucleic acid molecules consisting of thebase sequences of SEQ ID NOs: 4 to 6 shown below were obtained, and asthe binding nucleic acid molecule that binds to lysozyme, bindingnucleic acid molecules consisting of the base sequences of SEQ ID NOs: 7to 9 shown below were obtained. In the base sequences of SEQ ID NOs: 4to 9 below, each underlined A is MK4.

BDN4A-binding nucleic acid molecule 1 (SEQ ID NO: 4)5′-GGTTACACGAGCCGCACATTTCTATTTTTACGGGGTATAGTTCTCTGAGGAGGAGTTCCCAGGCGAAGTTGTTATC-3′BDN4A-binding nucleic acid molecule 2 (SEQ ID NO: 5)5′-CGAGCCGCACATTTCTATTTTTACGGGGTATAGTTCTCTGAGGAGGAGTTCCCAGGCGAAGTTGTTATC-3′ BDN4A-binding nucleic acid molecule 3(SEQ ID NO: 6) 5′-GGTTACACGAGCCGCACATTTCACCGTGATAGTTCTCTGAGGAGGACTTCTAGAGTTCCCAGGCGAAGTTGTTATC-3′Lysozyme-binding nucleic acid molecule 1 (SEQ ID NO: 7)5′-GGTTACACGAGCCGCACATTTCTAACGGGAACTTCAACCCATACAGTCTTTTGAGTTCCCAGGCGAAGTTGTTATC-3′Lysozyme-binding nucleic acid molecule 2 (SEQ ID NO: 8)5′-CGAGCCGCACATTTCTAACGGGAACTTCAACCCATACAGTCTTT TGAGTTCCC-3′Lysozyme-binding nucleic acid molecule 3 (SEQ ID NO: 9)5′-GGTTACACGAGCCGCACATTTCTTTACTCCGGAACCCATACAGTCTTTTCCGGAGTTCCCAGGCGAAGTTGTTATC-3′

(2) Examination of Binding by SPR

The binding between each type of the BDN4A-binding nucleic acidmolecules and BDN4A and the binding between each type of thelysozyme-binding nucleic acid molecules and lysozyme were measured inthe same manner as in the above item (2) in Example 2, except that theBDN4A-binding nucleic acid molecules or lysozyme-binding nucleic acidmolecules having a 20-mer poly(dT) added to their 3′ ends were used asthe ligand 2 and each of the following proteins was used as the analyte.As controls, the binding was examined in the same manner except thatα-amylase and sIgA were used as the analytes.

-   -   β-defensin 4A: P-Defensin 4A, Human (manufactured by Novoprotein        Scientific Inc., Catalog number: #C127)    -   Human lysozyme: Recombinant Human Lysozyme C (manufactured by        Novoprotein Scientific Inc., Catalog number: #P61626)

The results of measuring the binding between the respective types of theBDN4A-binding nucleic acid molecules and BDN4A are shown in FIGS. 5A to5C. The results of measuring the binding between the respective types ofthe lysozyme-binding nucleic acid molecules and the lysozyme are shownin FIGS. 6A to 6C.

FIGS. 5A to 5C are graphs showing the binding ability of the respectivetypes of the BDN4A-binding nucleic acid molecules to BDN4A. FIGS. 5A to5C show the results obtained regarding the BDN4A-binding nucleic acidmolecules 1 to 3, respectively. In each of FIGS. 5A to 5C, thehorizontal axis indicates the time elapsed after the injection of theligand, and the vertical axis indicates the relative value of thebinding force (RU). As can be seen in FIGS. 5A to 5C, the respectivetypes of the BDN4A-binding nucleic acid molecules did not bind to theamylase or sIgA and bound to BDN4A.

Next, FIGS. 6A to 6C are graphs showing the binding ability of therespective types of the lysozyme-binding nucleic acid molecules to thelysozyme. FIGS. 6A to 6C show the results obtained regarding thelysozyme-binding nucleic acid molecules 1 to 3, respectively. In each ofFIGS. 6A to 6C, the horizontal axis indicates the time elapsed after theinjection of the ligand, and the vertical axis indicates the relativevalue (RU) of the binding force. As can be seen in FIGS. 6A to 6C, thelysozyme-binding nucleic acid molecules did not bind to the amylase orsIgA and bound to the lysozyme.

From these results, it was found that a binding nucleic acid moleculethat binds to BDN4A and a binding nucleic acid molecule that binds tolysozyme can be obtained using MK4, which is the nucleoside derivativeof the present invention.

(3) Examination of Binding Force

The relative value (RU) of the binding force was measured in the samemanner as in the above item (2) in Example 3, except that theBDN4A-binding nucleic acid molecules having a 20-mer poly(T) added totheir 3′ ends were used as the ligand 2 and that the concentration ofBDN4A as the analyte was set to 25, 50, 100, 200, or 400 nmol/L. Also,the relative value (RU) of the binding force was measured in the samemanner as in the item (2) in Example 3, except that each type of thelysozyme-binding nucleic acid molecules having a 20-mer poly(T) added totheir 3′ ends were used as the ligand 2 and that the concentration ofthe lysozyme as the analyte was set to 12.5, 25, 50, 100, or 200 nmol/L.Then, on the basis of the relative values (RU) of the binding forcemeasured in the above, the dissociation constant between each type ofthe BDN4A-binding nucleic acid molecules and BDN4A and the dissociationconstant between each type of the lysozyme-binding nucleic acidmolecules and the lysozyme were calculated. The results obtained areshown in Table 1 below.

TABLE 1 Dissociation Nucleic acid molecule name constant (nM)BDN4A-binding nucleic acid molecule 1 6.52 BDN4A-binding nucleic acidmolecule 2 8.07 BDN4A-binding nucleic acid molecule 3 48.2Lysozyme-binding nucleic acid molecule 1 1.18 Lysozyme-binding nucleicacid molecule 2 1.03 Lysozyme-binding nucleic acid molecule 3 1.83

As can be seen in Table 1 above, it was found that these binding nucleicacid molecules all have excellent binding ability to the targets.

(4) Examination of Binding by Pull-Down Assay

Beads carrying BDN4A-binding nucleic acid molecules bound thereto (alsoreferred to as “bound beads C” hereinafter) and beads carryinglysozyme-binding nucleic acid molecules bound thereto (also referred toas “bound beads D” hereinafter) were prepared by bringing BDN 4A-bindingnucleic acid molecules 1 and lysozyme-binding nucleic acid molecules 1with their 5′ ends modified with biotin into contact with theabove-described streptavidin-modified beads, respectively. Next,SDS-PAGE was performed, and gel was imaged in the same manner as in theitem (5) in Example 2, except that the bound beads C and D were eachmixed with a SB buffer containing 90 (v/v)% saliva (saliva sample) or aSB buffer containing BDN4A or lysozyme (target sample). Further, ascontrol 1 and control 2, imaging was performed in the same manner exceptthat, in control 1, the following control nucleic acid molecules 2 withtheir 5′ ends modified with biotin were used and, in control 2, onlyBDN4A or only lysozyme was used.

Control nucleic acid molecule 2 (SEQ ID NO: 10)5′-GGTAACCGCCCTGTCTTGATAAC-3′

Next, the results obtained when the bound beads C were used are shown inFIG. 7. FIG. 7 is a photograph showing the results of the pull-downassay using the bound beads C. In FIG. 7, the numerical values on theleft side of the photograph indicate molecular weights, and therespective lanes are, from the left, lane M (marker), lane 4 (targetsample), lane C1 (control 1), lane hBDN (control 2), lane M (marker),lane 8 (saliva sample), lane C2 (control 1), and lane hBDN (control 2).As can be seen in FIG. 7, in the lane showing the result of control 1,no band was observed at the same position (about 10 kDa) as in the laneshowing the result of control 2, whereas, in the lanes showing theresults obtained when the target sample and the saliva sample were used,bands were observed at the same electrophoretic mobility position as inthe lane showing the result of control 2, as indicated with the arrowsin FIG. 7. In other words, binding of the BDN4A-binding nucleic acidmolecules 1 to BDN4A was observed. From these results, it was found thatthe BDN4A-binding nucleic acid molecules bind to BDN4A.

Next, the results obtained when the bound beads D were used are shown inFIGS. 8A and 8B. FIGS. 8A and 8B are photographs showing the results ofthe pull-down assay. FIG. 8A shows the result obtained when the targetsample was used and FIG. 8B shows the result obtained when the salivasample was used. In FIG. 8A, the numerical values on the left side ofthe photograph indicate molecular weights, and the respective lanes are,from the left, lane M (marker), lane 2 (target sample), lane C1 (control1), lane hLys (control 2), and lane M (marker). In FIG. 8B, therespective lanes are, from the left, lane M (marker), lane 6 (salivasample), lane C1 (control 1), lane hLys (control 2), and lane M(marker). As can be seen in FIG. 8, in the lane showing the result ofcontrol 1, no band was observed at the same position (about 15 kDa) asin the lane showing the result of control 2, whereas, in the lanesshowing the results obtained when the target sample and the salivasample were used, bands were observed at the same electrophoreticmobility position as in the lane showing the result of control 2, asindicated with the arrows in FIG. 8. In other words, binding of thelysozyme-binding nucleic acid molecules 1 to the lysozyme was observed.From these results, it was found that the lysozyme-binding nucleic acidmolecules bind to lysozyme.

Example 4

The present example examined whether binding nucleic acid molecules thatbind to human α-amylase can be obtained using MK4.

(1) Binding Nucleic Acid Molecule

Binding nucleic acid molecules that bind to human α-amylase wereobtained in the same manner as in the item (1) in Example 2, exceptthat, instead of sIgA as the target, the above-described human α-amylasewas used.

As a result, as the binding nucleic acid molecules that bind toα-amylase, binding nucleic acid molecules consisting of the basesequences of SEQ ID NOs: 11 to 16 shown below were obtained. In the basesequences of SEQ ID NOs: 11 to 16 below, each underlined A is MK4.

α-amylase-binding nucleic acid molecule 2 (SEQ ID NO: 11)5′-GGTTTGGACGCAATCTCCCTAATCTAGTGACGAAAATGTACGAG GGGGTCATTTGAAACTA-3′α-amylase-binding nucleic acid molecule 3 (SEQ ID NO: 12)5′-GCAATCTCCCTAATCTAGTGACGAAAATGTACGAGGGGGTCATT TGAAACTA-3′α-amylase-binding nucleic acid molecule 4 (SEQ ID NO: 13)5′-GGTTTGGACGCAATCTCCCTAATCAGACTATTATTTCAAGTACGTGGGGGTCTTGAAACTACAATGGGCGGGCTTATC-3′α-amylase-binding nucleic acid molecule 5 (SEQ ID NO: 14)5′-GGTTTGGACGCAATCTCCCTAATCTAAAGTTTCTAAACGATGTGGCGGCATTCAGAAACTACAATGGGCGGGCTTATC-3′α-amylase-binding nucleic acid molecule 6 (SEQ ID NO: 15)5′-GGTTTGGACGCAATCTCCCTAATCTAAAGTTTCTAAACGATGTG GCGGCATTCAGAAACT-3′α-amylase-binding nucleic acid molecule 7 (SEQ ID NO: 16)5′-GCAATCTCCCTAATCTAAAGTTTCTAAACGATGTGGCGGCATTC AGAAACT-3′

(2) Examination of Binding by SPR

The binding between each type of the α-amylase-binding nucleic acidmolecules and the α-amylase was measured in the same manner as formeasuring the binding of the α-amylase-binding nucleic acid molecules 1in the above item (2) in Example 2. As controls, the binding wasexamined in the same manner except that CgA and BSA were used as theanalytes. The results obtained are shown in FIGS. 9A to 9F.

FIGS. 9A to 9F are graphs showing the binding ability of the respectivetypes of the α-amylase-binding nucleic acid molecules to the α-amylase.In FIGS. 9A to 9F show the results obtained regarding theα-amylase-binding nucleic acid molecules 2 to 7, respectively. In eachof FIGS. 9A to 9F, the horizontal axis indicates the time elapsed afterthe injection of the ligand, and the vertical axis indicates therelative value (RU) of the binding force. As can be seen in FIGS. 9A to9F, the respective types of the α-amylase-binding nucleic acid moleculesdid not bind to CgA or BSA and bound to the α-amylase.

(3) Examination of Binding Force

The relative value (RU) of the binding force was measured in the samemanner as in the item (2) in Example 4, except that each type of theα-amylase-binding nucleic acid molecules having a 20-mer poly(T) addedto their 3′ ends were used as the ligand 2, and the concentration of theα-amylase as the analyte was set to 5, 10, 20, 40, or 80 nmol/L. Then,on the basis of the relative values (RU) of the binding force measuredin the above, the dissociation constant between each type of theα-amylase-binding nucleic acid molecules and the α-amylase wascalculated. As a result, the dissociation constants between theα-amylase-binding nucleic acid molecules 2 to 7 and the α-amylase were6.91, 7.75, 5.18, 13.2, 11.5, and 11.1 nM, respectively. These resultsdemonstrate that all of the binding nucleic acid molecules haveexcellent binding ability to the targets.

(4) Examination of Binding by Capillary Electrophoresis

The binding was measured in the same manner as in the item (4) inExample 2, except that, in addition to the α-amylase-binding nucleicacid molecules 1, the α-amylase-binding nucleic acid molecules 5 wereused. As a control, the measurement was performed in the same manner,except that the α-amylase was not added as the target.

The results obtained are shown in FIG. 10. FIG. 10 is a photographshowing the results of capillary electrophoresis. In FIG. 10, theelectrophoresis time is shown on the left side of the photograph, andthe respective lanes show, from the left, the results obtained regardingthe α-amylase-binding nucleic acid molecules 1 in the control (withoutα-amylase) and in the presence of the α-amylase, and the resultsobtained regarding the α-amylase-binding nucleic acid molecules 5 in thecontrol (without α-amylase) and in the presence of the α-amylase. As canbe seen in FIG. 10, regarding the respective types of theα-amylase-binding nucleic acid molecules, the electrophoresis time inthe presence of the α-amylase was longer than that in the controlwithout α-amylase. From these results, it was found that theα-amylase-binding nucleic acid molecules 1 and 5 bind to α-amylase.

(5) Examination of Binding by Pull-Down Assay

Beads 5 carrying α-amylase-binding nucleic acid molecules bound thereto(also referred to as “bound beads A5” hereinafter) were prepared bybringing the α-amylase-binding nucleic acid molecules 5 with their 5′ends modified with biotin into contact with the above-describedstreptavidin-modified beads. Then, imaging was performed in the samemanner as in the item (5) in Example 2, except that the bound beads A5were used in addition to the bound beads A1 and that a target samplecontaining α-amylase was used as the sample. Further, as control 1 andcontrol 2, imaging was performed in the same manner except that, incontrol 1, nucleic acid molecules that do not bind to α-amylase withtheir 5′ ends modified with biotin were used (the control nucleic acidmolecules 1) and, in control 2, only α-amylase was used.

The results obtained are shown in FIG. 11. FIG. 11 is a photographshowing the results of the pull-down assay using the bound beads A1 andA5. In FIG. 11, the numerical values on the left side of the photographindicate molecular weights, and the respective lanes show, from theleft, the results obtained regarding the molecular weight marker (M),the bound beads A1 (1), the bound beads A5 (2), control 1 (C1), andcontrol 2 (AMY). As can be seen in FIG. 11, in the lane showing theresult of control 1, no band was observed at the same position (about 50kDa) as in the lane showing the result of control 2, whereas, in thelanes showing the results regarding the bound beads A1 and A5, bandswere observed at the same electrophoretic mobility position as in thelane showing the result of control 2. In other words, the binding of theα-amylase-binding nucleic acid molecules 1 and 5 to α-amylase wasobserved. From these results, it was found that the α-amylase-bindingnucleic acid molecules 1 and 5 bind to α-amylase.

Example 5

The present example examined whether binding nucleic acid molecules thatbind to human LDHS and binding nucleic acid molecules that bind to humanIL-6 can be obtained using MK4.

(1) Binding Nucleic Acid Molecule

Binding nucleic acid molecules that bind to human LDHS and bindingnucleic acid molecules that bind to human IL-6 were obtained in the samemanner as in the item (1) in Example 2, except that, instead of sIgA asthe target, human LDHS (manufactured by Meridian Life Science, Inc.,Catalog number: #A38558H-100) and human IL-6 (manufactured by MPBiomedicals, LLC-Cappel Products, Catalog number: #55905) were used,respectively.

As a result, as the binding nucleic acid molecules that bind to LDHS,binding nucleic acid molecules consisting of the base sequences of SEQID NOs: 17 to 20 shown below were obtained, and as the binding nucleicacid molecules that bind to IL-6, binding nucleic acid moleculesconsisting of the base sequences of SEQ ID NOs: 21 and 22 shown belowwere obtained. In the base sequences of SEQ ID NOs: 17 to 22 shownbelow, each underlined A is MK4.

LDH5-binding nucleic acid molecule 1 (SEQ ID NO: 17)5′-GGAATTGACACCTCGCCGTTTATGCTGCTGGCTCGTGAGACGGATATCAGGTCTCCTAAGGCTGGCTGGCTACTATAC-3′LDH5-binding nucleic acid molecule 2 (SEQ ID NO: 18)5′-GGAATTGACACCTCGCCGTTTATGAGAGGGAGATCATCTCTCTGGCGGACACAACCTAAGGCTGGCTGGCTACTATAC-3′LDH5-binding nucleic acid molecule 3 (SEQ ID NO: 19)5′-ACCTCGCCGTTTATGCTGCTGGCTCGTGAGACGGATATCAGGTC TCCTAAGGCTGGC-3′LDH5-binding nucleic acid molecule 4 (SEQ ID NO: 20)5′-TGCTGCTGGCTCGTGAGACGGATATCAGGTCTCCTAAGGCTGG C-3′IL-6-binding nucleic acid molecule 1 (SEQ ID NO: 21)5′-GGAATTGACACCTCGCCGTTTATGAGTTCAATGGTATTGTATCGACTCTTCTCGCCTAAGGCTGGCTGGCTACTATAC-3′IL-6-binding nucleic acid molecule 2 (SEQ ID NO: 22)5′-ACCTCGCCGTTTATGAGTTCAATGGTATTGTATCGACTCTTCT C-3′

(2) Examination of binding by SPR

The binding between the LDH5-binding nucleic acid molecules and LDH5 andthe binding between the IL-6-binding nucleic acid molecules and IL-6were measured in the same manner as in the item (2) in Example 2, exceptthat the LDH5-binding nucleic acid molecules or the IL-6-binding nucleicacid molecules having a 20-mer poly(dT) added to their 3′ ends were usedas the ligand 2 and each of the following proteins was used as theanalyte. As controls, the binding was examined in the same manner exceptthat α-amylase and sIgA were used as the analytes.

-   -   LDH5: Lactate Dehydrogenase 5, Human (manufactured by Meridian        Life Science, Inc., Catalogue number: #A38558H-100)    -   CgA: Recombinant full length Human Chromogranin A (manufactured        by Creative BioMart, Catalogue number: #CHGA 26904TH)    -   IL-6: IL-6, manufactured by Human, Recombinant (manufactured by        PeproTech, Catalogue number: #200-06)    -   Amylase: α-amylase (manufactured by Lee Biosolutions, Catalogue        number: #120-10)    -   sIgA: IgA (Secretory), Human (manufactured by MP Biomedicals,        LLC-Cappel Products, Catalogue number: #55905)

The results of measuring the binding between the respective types of theLDH5-binding nucleic acid molecule and LDH5 are shown in FIG. 12, andthe results of measuring the binding between the respective types ofIL-6-binding nucleic acid molecules and IL-6 are shown in FIG. 13.

FIGS. 12A to 12D are graphs showing the binding ability of therespective types of the LDH5-binding nucleic acid molecules to LDH5.FIGS. 12A to 12D show the results obtained regarding the LDH5-bindingnucleic acid molecules 1 to 4, respectively. In each of FIGS. 12A to12D, the horizontal axis indicates the time elapsed after the injectionof the ligand, and the vertical axis indicates the relative value (RU)of the binding force. As can be seen in FIGS. 12A to 12D, the respectivetypes of the LDH5-binding nucleic acid molecules did not bind to theamylase or sIgA and bound to LDH5.

FIGS. 13A and 13B are graphs showing the binding ability of therespective types of IL-6-binding nucleic acid molecules to IL-6. FIGS.13A and 13B show the results obtained regarding the IL-6-binding nucleicacid molecules 1 and 2, respectively. In each of FIGS. 13A and 13B, thehorizontal axis indicates the time elapsed after the injection of theligand, and the vertical axis indicates the relative value (RU) of thebinding force. As can be seen in FIGS. 13A and 13B, the respective typesof IL-6-binding nucleic acid molecules did not bind to the amylase orsIgA and bound to IL-6.

Next, the binding ability of each type of the binding nucleic acidmolecules was examined on the basis of the amount of each type of thebinding nucleic acid molecules immobilized on a measurement chip and thebinding amount of each type of the binding nucleic acid molecules to thetarget. Specifically, the signal intensity (RU) after the injection ofthe ligand 2 was measured, and the measured value, which corresponds toa signal indicating the amount of each type of the binding nucleic acidmolecules immobilized on the measurement chip, was regarded as an“nucleic acid molecule immobilization measured value (A)”. Further,signal intensity measurement was performed concurrently with injectionof the analyte and washing with a buffer. With 0 seconds being the startof the injection, the mean value of signal intensities from 115 secondsto 125 seconds was determined. This result, which corresponds to asignal indicating the binding amount between each type of the bindingnucleic acid molecules and the target, was regarded as a “target bindingmeasured value (B)”. Then, the value (B/A) obtained by dividing thetarget binding measured value (B) by the nucleic acid moleculeimmobilization measured value (A) was determined as a relative value(Relative Unit), and the thus-obtained value was regarded as the bindingability. As controls, the binding ability was determined in the samemanner, except that α-amylase and sIgA were used as the analytes.

The results obtained regarding the respective types of the LDH5-bindingnucleic acid molecules are shown in FIG. 14, and the results obtainedregarding the respective types of IL-6-binding nucleic acid moleculesare shown in FIG. 15.

FIG. 14 is a graph showing the relative values (Relative Units) of thebinding amounts of the respective types of the LDH5-binding nucleic acidmolecules to LDH5. In FIG. 14, the horizontal axis indicates the type ofthe LDH5-binding nucleic acid molecules, and the vertical axis indicatesthe relative value. As can be seen in FIG. 14, no binding was observedin the control in which α-amylase or sIgA was used. In contrast, all thetypes of the LDH5-binding nucleic acid molecules bound to LDH5.

FIG. 15 is a graph showing the relative values of the binding amounts ofthe respective types of the IL-6-binding nucleic acid molecules to IL-6.In FIG. 15, the horizontal axis indicates the types of the IL-6-bindingnucleic acid molecules, and the vertical axis indicates the relativevalue. As can be seen in FIG. 15, no binding was observed in the controlin which α-amylase or sIgA was used. In contrast, all the types of theIL-6-binding nucleic acid molecules bound to IL-6.

From these results, it was found that a binding nucleic acid moleculethat binds to LDH5 and a binding nucleic acid molecule that binds toIL-6 can be obtained using MK4, which is the nucleoside derivativeaccording to the present invention.

(3) Examination of Binding Force

The relative value (RU) of the binding force was measured in the samemanner as in the item (2) in Example 5, except that each type of theLDH5-binding nucleic acid molecules having a 20-mer poly(T) added totheir 3′ ends were used as the ligand 2 and that the concentration ofLDH5 as the analyte was set to 1.25, 2.5, 5, 10, or 20 nmol/L or to6.25, 12.5, 25, 50, or 100 nmol/L. Also, the relative value of thebinding force (RU) was measured in the same manner as in the item (2) inExample 5, except that each type of the IL-6-binding nucleic acidmolecules having a 20-mer poly(T) added to their 3′ ends were used asthe ligand 2 and that the concentration of IL-6 as the analyte was setto 6.25, 12.5, 25, 50, or 100 nmol/L or to 12.5, 25, 50, 100, or 200nmol/L. Then, on the basis of the relative values of the binding force(RU) measured in the above, the dissociation constant between each typeof the LDH5-binding nucleic acid molecules and LDH5 and the dissociationconstant between each type of the IL-6-binding nucleic acid moleculesand IL-6 were calculated. The results obtained are shown in Table 2below.

TABLE 2 Dissociation Nucleic acid molecule name constant (nM)LDH5-binding nucleic acid molecule 1 1.68 LDH5-binding nucleic acidmolecule 2 0.49 LDH5-binding nucleic acid molecule 3 0.53 LDH5-bindingnucleic acid molecule 4 0.23 LDH5-binding nucleic acid molecule 5 2.54LDH5-binding nucleic acid molecule 6 1.38

As can be seen in Table 2 above, it was found that these binding nucleicacid molecules all have excellent binding ability to the targets. Inparticular, the LDH5-binding nucleic acid molecules 2 to 4 exhibitedparticularly excellent binding ability to the target.

(4) Examination of Binding by Pull-Down Assay

Beads carrying LDH5-binding nucleic acid molecules bound thereto (alsoreferred to as “bound beads L” hereinafter) were prepared by bringingthe LDH5-binding nucleic acid molecules 1 with their 5′ ends modifiedwith biotin into contact with the above-described streptavidin-modifiedbeads. Next, SDS-PAGE was performed and gel was imaged in the samemanner as in the item (5) in Example 2, except that the bound beads Lwere mixed with a SB buffer containing 90 (v/v)% saliva (saliva sample)or a SB buffer containing LDH5 (target sample). Further, as control 1and control 2, imaging was performed in the same manner except that, incontrol 1, the following control nucleic acid molecules 3 with their 5′ends modified with biotin were used and, in control 2, only LDH5 wasused.

Control nucleic acid molecule 3 (SEQ ID NO: 23)5′-GGAATTGACACCTCGCCGTTTATG-3′

Next, the results obtained when the bound beads L were used are shown inFIG. 16. FIG. 16 is a photograph showing the results of the pull-downassay using the bound beads L. In FIG. 16, the numerical values on theleft side of the photograph indicate molecular weights, and therespective lanes are, from the left, lane M (marker), lane 1 (targetsample), lane C1 (control 1), lane LDH5 (control 2), lane M (marker),lane 2 (saliva sample), and lane C2 (control 1). As can be seen in FIG.16, in the lane showing the result of control 1, no band was observed atthe same position (about 35 kDa) as in the lane showing the result ofcontrol 2, whereas, in the lanes showing the results obtained when thetarget sample and the saliva sample were used, bands were observed atthe same electrophoretic mobility position as in the lane showing theresult of control 2, as indicated with the arrows in FIG. 16. In otherwords, binding of the LDH5-binding nucleic acid molecules 1 to LDH5 wasobserved. From these results, it was found that the LDH5-binding nucleicacid molecules bind to LDH5.

Although the present invention is described above with reference toembodiments and examples, the present invention is not limited thereto.Various modifications can be made within the scope of the presentinvention which can be understood by those skilled in the art.

The present application is based upon and claims the benefit of priorityfrom Japanese patent application No. 2016-180894, filed on September 15,2016 and International patent application No. PCT/JP2017/020065, filedon May 30, 2017, and the entire disclosure of which is incorporatedherein in its entirety by reference.

(Supplementary Notes)

Some or all of the above-described embodiments and examples may bedescribed, but are not limited to, as the following Supplementary Notes.

(Supplementary Note 1)

A nucleoside derivative or a salt thereof, represented by the followingchemical formula (1):

where in the chemical formula (1),

Su is an atomic group having a sugar skeleton at a nucleoside residue oran atomic group having a sugar phosphate skeleton at a nucleotideresidue, and may or may not have a protecting group,

L¹ and L² are each independently a straight-chain or branched, saturatedor unsaturated hydrocarbon group having 2 to 10 carbon atoms,

X¹ and X² are each independently an imino group (—NR¹—), an ether group(—O—), or a thioether group (—S—), and

the R¹ is a hydrogen atom or a straight-chain or branched, saturated orunsaturated hydrocarbon group having 2 to 10 carbon atoms.

(Supplementary Note 2)

The nucleoside derivative or a salt thereof according to SupplementaryNote 1, wherein the X¹ is an imino group (—NR¹—).

(Supplementary Note 3)

The nucleoside derivative or a salt thereof according to SupplementaryNote 1 or 2, wherein the X² is an imino group (—NR¹—).

(Supplementary Note 4)

The nucleoside derivative or a salt thereof according to SupplementaryNote 2 or 3, wherein the R¹ is a hydrogen atom.

(Supplementary Note 5)

The nucleoside derivative or a salt thereof according to any one ofSupplementary Notes 1 to 4, wherein the L¹ is a vinylene group(—CH═CH—).

(Supplementary Note 6) The nucleoside derivative or a salt thereofaccording to any one of Supplementary Notes 1 to 5, wherein the L² is anethylene group (—CH₂—CH₂—).

(Supplementary Note 7)

The nucleoside derivative or a salt thereof according to any one ofSupplementary Notes 1 to 6, wherein an atomic group having a sugarskeleton at the nucleoside residue or an atomic group having a sugarphosphate skeleton at the nucleotide residue is represented by thefollowing chemical formula (2):

where in the chemical formula (2),

R² is a hydrogen atom, a protecting group, or a group represented by thefollowing chemical formula (3),

R³ is a hydrogen atom, a protecting group, or a phosphoramidite group,

R⁴ is a hydrogen atom, a fluorine atom, a hydroxyl group, an aminogroup, or a mercapto group,

where in the chemical formula (3),

Y is an oxygen atom or a sulfur atom,

Z is a hydroxyl group or an imidazole group, and

m is an integer of 1 to 10.

(Supplementary Note 8)

The nucleoside derivative or a salt thereof according to any one ofSupplementary Notes 1 to 7, wherein the nucleoside derivativerepresented by the chemical formula (1) is a nucleoside derivativerepresented by the following chemical formula (4):

(Supplementary Note 9)

A polynucleotide synthesis reagent comprising a nucleotide derivative ora salt thereof that comprises the nucleoside derivative or a saltthereof according to any one of Supplementary Notes 1 to 8.

(Supplementary Note 10)

A method for producing a polynucleotide, comprising the step ofsynthesizing a polynucleotide using a nucleotide derivative or a saltthereof that comprises the nucleoside derivative or a salt thereofaccording to any one of Supplementary Notes 1 to 8.

(Supplementary Note 11)

A polynucleotide comprising, as a building block, a nucleotidederivative or a salt thereof that comprises the nucleoside derivative ora salt thereof according to any one of

Supplementary Notes 1 to 8.

(Supplementary Note 12)

The polynucleotide according to Supplementary Note 11, wherein thepolynucleotide is a binding nucleic acid molecule that binds to atarget.

(Supplementary Note 13)

The polynucleotide according to Supplementary Note 12, wherein thetarget is at least one selected from the group consisting of secretoryimmunoglobulin A, amylase, β-defensin 4A, lysozyme, lactatedehydrogenase (LDH) 5, and interleukin (IL)-6.

(Supplementary Note 14)

A method for producing a binding nucleic acid molecule, comprising thesteps of:

causing a candidate polynucleotide and a target to come into contactwith each other; and

selecting the candidate polynucleotide bound to the target as a bindingnucleic acid molecule that binds to the target, wherein

the candidate polynucleotide is the polynucleotide according to any oneof

Supplementary Notes 11 to 13.

(Supplementary Note 15)

The method according to Supplementary Note 14, wherein the target is atleast one selected from the group consisting of secretory immunoglobulinA, amylase, β-defensin 4A, lysozyme, lactate dehydrogenase (LDH) 5, andinterleukin (IL)-6.

(Supplementary Note 16)

An α-amylase-binding nucleic acid molecule comprising a polynucleotide(a):

(a) a polynucleotide (a1):

(a1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:1 and 11 to 16.

(Supplementary Note 17)

The α-amylase-binding nucleic acid molecule according to SupplementaryNote 16, wherein the α-amylase-binding nucleic acid molecule comprises amodified base, which is a base modified.

(Supplementary Note 18)

The α-amylase-binding nucleic acid molecule according to SupplementaryNote 17, wherein the modified base is a modified purine base, which is apurine base modified with a modifying group.

(Supplementary Note 19)

The α-amylase-binding nucleic acid molecule according to SupplementaryNote 18, wherein modifying group is an adenine residue.

(Supplementary Note 20)

The α-amylase-binding nucleic acid molecule according to any one ofSupplementary Notes 16 to 19, wherein the polynucleotide is DNA.

(Supplementary Note 21)

A method for analyzing α-amylase, comprising the step of:

causing a specimen and a nucleic acid molecule to come into contact witheach other to detect α-amylase in a specimen, wherein

the nucleic acid molecule is an α-amylase-binding nucleic acid moleculeaccording to any one of Supplementary Notes 16 to 20, and

in the detection, the nucleic acid molecule is caused to bind to theα-amylase in the specimen, and the α-amylase in the specimen is detectedby detecting the binding.

(Supplementary Note 22)

The method according to Supplementary Note 21, wherein the specimen isat least one selected from the group consisting of saliva, urine,plasma, and serum.

(Supplementary Note 23)

A β-defensin (BDN)4A-binding nucleic acid molecule comprising apolynucleotide (b):

(b) a polynucleotide (b1):

(b1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:4 to 6.

(Supplementary Note 24)

The BDN4A-binding nucleic acid molecule according to Supplementary Note23, wherein the BDN4A-binding nucleic acid molecule comprises a modifiedbase, which is a base modified.

(Supplementary Note 25)

The BDN4A-binding nucleic acid molecule according to Supplementary Note24, wherein the modified base is a modified purine base, which is apurine base modified with a modifying group.

(Supplementary Note 26)

The BDN4A-binding nucleic acid molecule according to Supplementary Note25, wherein the modifying group is an adenine residue.

(Supplementary Note 27)

The BDN4A-binding nucleic acid molecule according to any one ofSupplementary Notes 23 to 26, wherein the polynucleotide is DNA.

(Supplementary Note 28)

A method for analyzing BDN4A, comprising the step of: causing a specimenand a nucleic acid molecule to come into contact with each other todetect β-defensin (BDN)4A in the specimen, wherein

the nucleic acid molecule is the BDN4A binding nucleic acid moleculeaccording to any one of Supplementary Notes 23 to 27, and

in the detection step, the nucleic acid molecule is caused to bind tothe BDN4A in the specimen, and the BDN4A in the specimen is detected bydetecting the binding.

(Supplementary Note 29)

The method according to Supplementary Note 28, wherein the specimen isat least one selected from the group consisting of saliva, urine,plasma, and serum.

(Supplementary Note 30)

A lysozyme-binding nucleic acid molecule comprising a polynucleotide(l):

(l) a polynucleotide (l1):

(l1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:7 to 9.

(Supplementary Note 31)

The lysozyme-binding nucleic acid molecule according to SupplementaryNote 30, wherein the lysozyme-binding nucleic acid molecule comprises amodified base, which is a base modified.

(Supplementary Note 32)

The lysozyme-binding nucleic acid molecule according to SupplementaryNote 31, wherein the modified base is a modified purine base, which is apurine base modified with a modifying group.

(Supplementary Note 33)

The lysozyme-binding nucleic acid molecule according to SupplementaryNote 32, wherein the modifying group is an adenine residue.

(Supplementary Note 34)

The lysozyme-binding nucleic acid molecule according to any one ofSupplementary Notes 30 to 33, wherein the polynucleotide is DNA.

(Supplementary Note 35)

A method for analyzing lysozyme, comprising the step of:

causing a specimen and a nucleic acid molecule to come into contact witheach other to detect lysozyme in the specimen, wherein

the nucleic acid molecule is the lysozyme-binding nucleic acid moleculeaccording to any one of Supplementary Notes 30 to 34, and

in the detection, the nucleic acid molecule is caused to bind to thelysozyme in the specimen, and the lysozyme in the specimen is detectedby detecting the binding.

(Supplementary Note 36) The method according to Supplementary Note 35,wherein the specimen is at least one selected from the group consistingof saliva, urine, plasma, and serum.

(Supplementary Note 37)

A lactate dehydrogenase (LDH)5-binding nucleic acid molecule comprisinga polynucleotide (d):

(d) a polynucleotide (d1):

(d1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:17 to 20.

(Supplementary Note 38)

The LDHS-binding nucleic acid molecule according to Supplementary Note37, wherein the LDHS-binding nucleic acid molecule comprises a modifiedbase, which is a base modified.

(Supplementary Note 39)

The LDHS-binding nucleic acid molecule according to Supplementary Note38, wherein the modified base is a modified thymine, which is a thyminebase modified with a modifying group.

(Supplementary Note 40)

The LDHS-binding nucleic acid molecule according to Supplementary Note39, wherein the modifying group is an adenine residue.

(Supplementary Note 41)

The LDHS-binding nucleic acid molecule according to any one ofSupplementary Notes 37 to 40, wherein the polynucleotide is DNA.

(Supplementary Note 42)

A method for analyzing LDH5, comprising the step of:

causing a specimen and a nucleic acid molecule to come into contact witheach other to detect lactate dehydrogenase (LDH)5 in the specimen,wherein

the nucleic acid molecule is the LDH5-binding nucleic acid moleculeaccording to any one of Supplementary Notes 37 to 41, and

in the detection step, the nucleic acid molecule is caused to bind tothe LDH5 in the specimen, and the LDH5 in the specimen is detected bydetecting the binding.

(Supplementary Note 43)

The method according to Supplementary Note 42, wherein the specimen isat least one selected from the group consisting of saliva, urine,plasma, and serum.

(Supplementary Note 44)

An interleukin (IL)-6 binding nucleic acid molecule comprising apolynucleotide (i):

(i) a polynucleotide (i1):

(i1) a polynucleotide consisting of any of base sequences of SEQ ID NOs:21 and 22.

(Supplementary Note 45)

The IL-6-binding nucleic acid molecule according to Supplementary Note44, wherein the IL-6-binding nucleic acid molecule comprises a modifiedbase, which is a base modified.

(Supplementary Note 46)

The IL-6-binding nucleic acid molecule according to Supplementary Note45, wherein the modified base is a modified thymine, which is a thyminebase modified with a modifying group.

(Supplementary Note 47)

The IL-6-binding nucleic acid molecule according to Supplementary Note46, wherein the modifying group is an adenine residue.

(Supplementary Note 48)

The IL-6-binding nucleic acid molecule according to any one ofSupplementary Notes 44 to 47, wherein the polynucleotide is DNA.

(Supplementary Note 49)

A method for analyzing IL-6, comprising the step of:

causing a specimen and a nucleic acid molecule to come into contact witheach other to detect interleukin (IL)-6 in the specimen, wherein

the nucleic acid molecule is the IL-6-binding nucleic acid moleculeaccording to any one of Supplementary Notes 44 to 48, and in thedetection step, the nucleic acid molecule is caused to bind to the IL-6in the specimen, and the IL-6 in the specimen is detected by detectingthe binding.

(Supplementary Note 50)

The method according to Supplementary Note 49, wherein the specimen isat least one selected from the group consisting of saliva, urine,plasma, and serum.

INDUSTRIAL APPLICABILITY

The present invention can provide a novel nucleoside derivative or asalt thereof. Further, the nucleoside derivative of the presentinvention has two purine ring-like structures. The nucleoside derivativeof the present invention thus has, for example, a relatively largernumber of atoms capable of interacting within or between molecules thana nucleoside derivative having one purine ring-like structure. Thebinding nucleic acid molecule including the nucleoside derivative of thepresent invention therefore has an improved binding ability to a target,for example, compared to a nucleoside derivative having one purinering-like structure. Thus, with the nucleoside derivative of the presentinvention, a binding nucleic acid molecule that exhibits excellentbinding ability to a target can be produced, for example. Accordingly,the present invention is really useful, for example, in the fields ofanalysis, medicine, life science, and the like.

-   [Sequence Listing] TF16066WO2_ST25.txt

1. A nucleoside derivative or a salt thereof, represented by thefollowing chemical formula (1):

where in the chemical formula (1), Su is an atomic group having a sugarskeleton at a nucleoside residue or an atomic group having a sugarphosphate skeleton at a nucleotide residue, and may or may not have aprotecting group, L¹ and L² are each independently a straight-chain orbranched, saturated or unsaturated hydrocarbon group having 2 to 10carbon atoms, X¹ and X² are each independently an imino group (—NR¹—),an ether group (—O—), or a thioether group (—S—), and the R¹ is ahydrogen atom or a straight-chain or branched, saturated or unsaturatedhydrocarbon group having 2 to 10 carbon atoms.
 2. The nucleosidederivative or a salt thereof according to claim 1, wherein the X¹ is animino group (—NR¹—).
 3. The nucleoside derivative or a salt thereofaccording to claim 1, wherein the X² is an imino group (—NR¹—).
 4. Thenucleoside derivative or a salt thereof according to claim 2, whereinthe R¹ is a hydrogen atom.
 5. The nucleoside derivative or a saltthereof according to claim 1, wherein the L¹ is a vinylene group(—CH═CH—).
 6. The nucleoside derivative or a salt thereof according toclaim 1, wherein the L² is an ethylene group (—CH₂—CH₂—).
 7. Thenucleoside derivative or a salt thereof according to claim 1, whereinthe atomic group having a sugar skeleton at a nucleoside residue or theatomic group having a sugar phosphate skeleton at a nucleotide residueis represented by the following chemical formula (2):

where in the chemical formula (2), R² is a hydrogen atom, a protectinggroup, or a group represented by the following chemical formula (3), R³is a hydrogen atom, a protecting group, or a phosphoramidite group, R⁴is a hydrogen atom, a fluorine atom, a hydroxyl group, an amino group,or a mercapto group,

where in the chemical formula (3), Y is an oxygen atom or a sulfur atom,Z is a hydroxyl group or an imidazole group, and m is an integer of 1 to10.
 8. The nucleoside derivative or a salt thereof according to claim 1,wherein the nucleoside derivative represented by the chemical formula(1) is a nucleoside derivative represented by the following chemicalformula (4):


9. A polynucleotide synthesis reagent comprising a nucleotide derivativeor a salt thereof that comprises the nucleoside derivative or a saltthereof according to claim
 1. 10. A method for producing apolynucleotide, comprising the step of synthesizing a polynucleotideusing a nucleotide derivative or a salt thereof that comprises thenucleoside derivative or a salt thereof according to claim
 1. 11-15.(canceled)